AAV delivery of nucleobase editors

Abstract

To provide methods of forming a complete and functional Cas9 protein or nucleobase editor.SOLUTION: Provided herein are methods of delivering "split" Cas9 protein or nucleobase editors into a cell via a recombinant adeno-associated virus (rAAV). The Cas9 protein or the nucleobase editor is split into two sections, each fused with a part of an intein system (e.g., intein-N and intein-C encoded by dnaEn and dnaEc, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Recombinant AAV vectors and particles for the delivery of the split Cas9 protein or nucleobase editor, and methods of using such AAV vectors and particles are also provided.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. USSN 62 / 408,575, filed October 14, 2016, and USSN 62 / 475,780, filed March 23, 2017, each of which is incorporated herein by reference.

[0002] Government support This invention was made with government support under grant numbers R35 GM118062 and R01 EB022376 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Precise genome targeting techniques using the CRISPR / Cas9 system have recently been investigated for a wide range of applications, including gene therapy. A major limiting factor for the application of Cas9 and Cas9-based genome editing agents in gene therapy is the size of Cas9 (>4 kb), which prevents its efficient delivery via recombinant adeno-associated virus (rAAV). Summary of the Invention

[0004] Abstract Described herein are systems, compositions, kits, and methods for delivering a Cas9 protein or nucleobase editor to cells, for example, via a recombinant adeno-associated virus vector. Typically, the Cas9 protein or nucleobase editor is "split" into an N-terminal portion and a C-terminal portion. The N-terminal or C-terminal portion of the Cas9 protein or nucleobase editor may each be fused to a member of an intein system. When delivered and co-expressed into a cell on separate vectors (e.g., separate rAAV vectors), the resulting fusion proteins can be linked to form a complete and functional Cas9 protein or nucleobase editor (e.g., via intein-mediated protein splicing). Also provided is experimental testing of regulatory elements in delivery vectors for high expression levels of split Cas9 proteins or nucleobase editors.

[0005] Some aspects of the present disclosure provide compositions comprising: (i) a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.

[0006] In some embodiments, the N-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, corresponding to amino acids 1-573 or 1-637 of SEQ ID NO: 1. In some embodiments, the C-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, corresponding to amino acids 574-1368 or 638-1368 of SEQ ID NO: 1. In some embodiments, intein-N comprises the amino acid sequence set forth in SEQ ID NOs: 350-351 and 354-355. In some embodiments, intein-C comprises the amino acid sequence set forth in SEQ ID NOs: 352-353 and 356-357.

[0007] In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises nucleotides encoding a guide RNA (gRNA) operably linked to a promoter.

[0008] In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises a transcription terminator.In some embodiments, the transcription terminator is the transcription terminator from bGH gene, hGH gene, or SV40 gene.In some embodiments, the transcription terminator is the transcription terminator from bGH gene.

[0009] In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted 5' of the transcription terminator.

[0010] In some embodiments, the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of KRPAATKKAGQAKKKK (SEQ ID NO: 344), KKTELQTTNAENKTKKL (SEQ ID NO: 345), KRGINDRNFWRGENGRKTR (SEQ ID NO: 346), and RKSGKIAAIVVKRPRK (SEQ ID NO: 347). In some embodiments, the bipartite nuclear localization signal comprises the amino acid sequence set forth in SEQ ID NO: 344.

[0011] In some embodiments, the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a nucleobase-modifying enzyme fused to the N-terminus of the N-terminal portion of the Cas9 protein.

[0012] In some embodiments, the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), and wherein the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a nucleobase-modifying enzyme fused to the C-terminus of the C-terminal portion of the Cas9 protein.

[0013] In some embodiments, the nucleobase-modifying enzyme is a deaminase. In some embodiments, the deaminase is a cytosine deaminase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) fused to the 3' end of the second nucleotide sequence. In some embodiments, the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) at the 5' end of the first nucleotide sequence. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NOs: 299-302.

[0014] In some embodiments, the first nucleotide sequence and the second nucleotide sequence are on different vectors. In some embodiments, each of the different vectors is a recombinant adeno-associated virus (rAAV) genome. In some embodiments, each vector is packaged in an rAAV particle.

[0015] Another aspect of the present disclosure provides compositions comprising: (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.

[0016] Cells comprising the compositions described herein are provided. In some embodiments, the N-terminal portion of the Cas9 protein and the C-terminal portion of the Cas9 protein are linked together to form a Cas9 protein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast cell, a plant cell, or a mammalian cell. In some embodiments, the cell is a human cell.

[0017] Further provided are kits comprising any of the compositions described herein.

[0018] Some aspects of the disclosure provide compositions comprising: (i) a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the nucleobase editor.

[0019] In some embodiments, intein-N comprises the amino acid sequence set forth in SEQ ID NOs: 350-351 and 354-355. In some embodiments, intein-C comprises the amino acid sequence set forth in SEQ ID NOs: 352-353 and 356-357. In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises nucleotides encoding a guide RNA (gRNA) operably linked to a promoter.

[0020] In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises a transcription terminator.In some embodiments, the transcription terminator is the transcription terminator from bGH gene, hGH gene, or SV40 gene.In some embodiments, the transcription terminator is from bGH gene.

[0021] In some embodiments, the first nucleotide sequence or the second nucleotide sequence further comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted 5' of the transcription terminator.

[0022] In some embodiments, the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal. In some embodiments, the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of KRPAATKKAGQAKKKK (SEQ ID NO: 344), KKTELQTTNAENKTKKL (SEQ ID NO: 345), KRGINDRNFWRGENGRKTR (SEQ ID NO: 346), and RKSGKIAAIVVKRPRK (SEQ ID NO: 347). In some embodiments, the bipartite nuclear localization signal comprises the amino acid sequence set forth in SEQ ID NO: 344.

[0023] In some embodiments, the nucleobase editor comprises a cytosine deaminase fused to the N-terminus of catalytically inactive Cas9 or Cas9 nickase. In some embodiments, the cytosine deaminase is selected from the group consisting of APOBEC1, APOBEC3, AID, and pmCDA1. In some embodiments, the nucleobase editor further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NOs: 299-302.

[0024] In some embodiments, the first nucleotide sequence and the second nucleotide sequence are on different vectors. In some embodiments, each of the different vectors is a recombinant adeno-associated virus (rAAV) genome. In some embodiments, the vector is packaged in an rAAV particle.

[0025] Another aspect of the disclosure provides a composition comprising: (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleic acid encoding an intein-C fused to the N-terminus of the C-terminal portion of the nucleobase editor.

[0026] Cells comprising any of the compositions described herein are provided. In some embodiments, the N-terminal portion of the nucleobase editor and the C-terminal portion of the nucleobase editor are linked together to form a nucleobase editor. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast cell, a plant cell, or a mammalian cell. In some embodiments, the cell is a human cell.

[0027] Further provided are kits comprising any of the compositions described herein.

[0028] Yet another aspect of the disclosure provides a method comprising contacting a cell with any of the compositions described herein, wherein the contacting results in delivery of a first nucleotide sequence and a second nucleotide sequence into the cell, and wherein the N-terminal portion of the nucleobase editor and the C-terminal portion of the nucleobase editor are linked to form the nucleobase editor.

[0029] Yet another aspect of the present disclosure provides a method comprising administering to a subject in need thereof a therapeutically effective amount of any of the compositions described herein. In some embodiments, the subject has a disease or disorder.

[0030] In some embodiments, the disease or disorder is selected from the group consisting of cystic fibrosis, phenylketonuria, epidermolytic keratosis (EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Tooth type 4J disease, neuroblastoma (NB), von Willebrand disease (vWD), congenital myotonia, hereditary renal amyloidosis, dilated cardiomyopathy, hereditary lymphedema, familial Alzheimer's disease, prion disease, chronic infantile neurological cutaneous and articular syndrome (CINCA), and desmin-related cardiomyopathy (DRM).

[0031] Details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as set forth below. Other features, objects, and advantages of the invention will be apparent from the definition, examples, drawings, and claims.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings constitute a part of this application and illustrate several aspects of the present invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0033] [Figure 1A]Figure 1A is a graph showing a "split nucleobase editor" for delivery into cells using a recombinant adeno-associated virus (rAAV) vector. Figure 1A is a schematic diagram of how the nucleobase editor is split into two parts. [Figure 1B] Figure IB is a graph showing "split nucleobase editors" for delivery into cells using recombinant adeno-associated virus (rAAV) vectors. Figure IB shows that the split nucleobase editor delivered by AAV can undergo protein splicing after expression of the two halves in cells to form a complete nucleobase editor with activity comparable to the nucleobase editor expressed as a whole. [Figure 1C] Figure 1C is a graph showing the formation of a complete nucleobase editor from the two halves via DnaE intein-mediated protein splicing.

[0034] [Figure 2] Figure 2 shows that U1118 cells were efficiently transfected with AAV2 containing a nucleic acid encoding mCherry. Various viral titers were tested (2.5 to 10 μl at 4.5 × 10 vg / ml*), and all resulted in efficient transfection of U118 cells. *vg / ml refers to viral genome-containing particles per microliter.

[0035] [Figure 3A] Figure 3A is a graph showing the results of high-throughput sequencing (HTS) of nucleobase editing by rAAV-delivered split nucleobase editors in U118 and HEK cells. A lipid-transfected nucleobase editor was used as a control. The PRNP locus was sequenced using an sgRNA targeting R37 in the PRNP gene. Figure 3A shows the HTS readout. [Figure 3B] Figure 3B is a graph showing the results of high-throughput sequencing (HTS) of nucleobase editing by rAAV-delivered split nucleobase editors in U118 and HEK cells. A lipid-transfected nucleobase editor was used as a control. The PRNP locus was sequenced using an sgRNA targeting R37 in the PRNP gene. Figure 3B summarizes the base editing results.

[0036] [Figure 4] Figure 4 is a graph showing the optimization of transcription terminators used in AAV constructs encoding split nucleobase editors. Transcription terminators of various sizes and origins were tested. The bGH transcription terminator is relatively short and efficiently terminates transcription compared to longer terminator sequences. Therefore, it was selected for use in downstream experiments.

[0037] [Figure 5]Figures 5A-5B are graphs showing the results of nucleobase editing by long-term (up to 15 days) transduction of AAVs encoding split nucleobase editors in mouse astrocytes expressing human ApoE4 cDNA. The target bases are in the codons for arginine 112 and arginine 158 in ApoE4, which are converted to cysteine ​​by base editing. Figure 5A shows that when mouse astrocytes are transduced with 10 vg, editing of arginine 158 increases over time, while editing of arginine 112 remains minimal. The nucleotide sequence 3' of the codon for arginine 158 sequence, with an adjacent NGG PAM, allows for high activity by SpCas9 (according to guide sequence GAAGCGCCTGGCAGTGTACC, SEQ ID NO: 348), while the nucleotide sequence 3' of the codon for arginine 112 contains an adjacent NAG PAM, which does not allow for high activity (according to guide sequence GACGTGCGCGGCCGCCTGGTG, SEQ ID NO: 349). Figure 5B shows cells transduced at 1010 vg with rAAV encoding mCherry (control).

[0038] [Figure 6] Figure 6 shows a schematic diagram of the optimization of a nuclear localization signal in an AAV construct encoding a split nucleobase editor. The nuclear localization signal controls nuclear import, which must occur for the reconstituted nucleobase editor to bind to genomic DNA as a prerequisite for editing, and is a potentially rate-limiting step in the process. This diagram demonstrates that the NLS (and optimization of the NLS) is important for the nucleobase editor to be imported into the nucleus.

[0039] [Figure 7] Figure 7 is a graph showing the results of base editing using various rAAV split nucleobase editor constructs containing various nuclear localization signals (NLS).

[0040] [Figure 8]8A-8B are graphs showing editing of the DNMT1 gene in dissociated mouse cortical neurons using AAVs encoding split nucleobase editors.

[0041] [Figure 9] 9A-9B are graphs showing editing of the DNMT1 gene in the mouse Neuro-2a cell line using either an AAV encoding a split nucleobase editor or DNA transfected with a lipid encoding a nucleobase editor. DETAILED DESCRIPTION OF THE INVENTION

[0042] definition As used in this specification and the claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent and a plurality of such agents.

[0043] As used herein, the term "Cas9," "Cas9 protein," or "Cas9 nuclease" refers to an RNA-guided nuclease, including Cas9 proteins (e.g., Cas9 nucleases from various bacterial species), fragments thereof, variants thereof (e.g., catalytically inactive Cas9 or Cas9 nickase), or fusion proteins (e.g., Cas9 fused to another protein domain). Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a nucleic acid that invades the target. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, accurate processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA acts as a guide for ribonuclease 3-assisted processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then cleaves the linear or circular dsDNA target complementary to the spacer by nucleotide excision. In nature, DNA binding and cleavage typically require both a protein and both RNAs. However, single-stranded guide RNAs ("sgRNAs," or simply "gNRAs") can be engineered to incorporate both crRNA and tracrRNA aspects into a single RNA species. Non-limiting examples of Cas9 proteins and their respective amino acid sequences are provided in Example 1.

[0044] Nuclease-inactivated Cas9 proteins can be interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9). Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., (2013) Cell. 28;152(5):1173-83, which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations in these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28;152(5):1173-83(2013)). Additional suitable nuclease-inactivated dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactivated Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., Nature Biotechnology. 2013;31(9):833-838, incorporated herein by reference).

[0045] In some embodiments, Cas9 nickases are used as part of a nucleobase editor. Cas9 nickases can cleave one strand of double-stranded DNA. Cas9 nickases can be generated by introducing inactivating mutations into either the HNH domain or the RuvC1 domain. For example, an inactivating mutation (D10A) can be introduced into the RuvC1 domain of S. pyogenes Cas9, while keeping the HNH domain active, i.e., leaving the residue at position 840 as a histidine. Such Cas9 variants can generate single-stranded DNA breaks (nicks) at specific locations based on the target sequence defined by the gRNA. Those skilled in the art can identify catalytic residues in the RuvC1 and HNH domains of any known Cas9 protein and introduce inactivating mutations to generate the corresponding dCas9 or nCas9.

[0046] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal portion (also referred to as the N-terminal half) and a C-terminal portion (also referred to as the C-terminal half) encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be combined (linked) to form a complete Cas9 protein. The Cas9 protein is known to consist of a bi-lobed structure connected by a disordered linker (e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, incorporated herein by reference). In some embodiments, the "split" occurs between the two lobes, resulting in two portions of the Cas9 protein, each containing one lobe.

[0047] An "intein" is a segment of a protein that can excise itself and link the remaining portion (an extein) via a peptide bond in a process known as protein splicing. Inteins are also referred to as "protein introns." The process by which an intein excises itself and links the remaining portion of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the inteins of a precursor protein (including the intein before intein-mediated protein splicing) are derived from two genes. Such inteins are referred to herein as split inteins. For example, in cyanobacteria, DnaE, ​​the catalytic subunit α of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene is referred to herein as "intein-N." The intein encoded by the dnaE-c gene is referred to herein as "intein-C."

[0048] Other intein systems can also be used.For example, synthetic inteins based on dnaE intein, Cfa-N and Cfa-C intein pairs have been described (for example, in Stevens et al., J Am Chem Soc. 2016 Feb 24;138(7):2162-5, which is incorporated herein by reference).Non-limiting examples of intein pairs that can be used according to the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (for example, those described in U.S. Patent 8,394,604, which is incorporated herein by reference).

[0049] Exemplary nucleotide and amino acid sequences of inteins are provided. DnaE intein-N, DNA: TGCCTGTCATACGAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGC AGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT (SEQ ID NO: 350) DnaE Intein-N, Protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN (SEQ ID NO: 351) DnaE intein-C, DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT (SEQ ID NO: 352) Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN (SEQ ID NO: 353) Cfa-N, DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAA CAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA (SEQ ID NO: 354) Cfa-N, protein: CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP (SEQ ID NO: 355) Cfa-C, DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC (SEQ ID NO: 356) Cfa-C, protein: MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 357)

[0050] For linking the N-terminal and C-terminal portions of split Cas9, intein-N and intein-C may be fused to the N-terminal and C-terminal portions of split Cas9, respectively. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split Cas9, i.e., forming the structure N-[N-terminal portion of split Cas9]-[intein-N]-C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split Cas9, i.e., forming the structure N-[intein-C]-[C-terminal portion of split Cas9]-C. Mechanisms of intein-mediated protein splicing for linking intein-fused proteins (e.g., split Cas9) are known in the art, as described, for example, in Shah et al., Chem Sci. 2014;5(1):446-461, incorporated herein by reference.

[0051] As used herein, a "nucleobase editor" refers to a protein that edits the bases of nucleotides. "Editing" refers to the conversion of one nucleobase to another (e.g., A to G, A to C, A to T, C to T, C to G, C to A, G to A, G to C, G to T, T to A, T to C, T to G). In some embodiments, a nucleobase editor is a macromolecule or macromolecular complex that primarily (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 99%, 99.9%, or 100%) effects the conversion of one nucleobase to another nucleobase in a polynucleic acid sequence (i.e., a transition or transversion) using a combination of 1) a nucleotide-, nucleoside-, or nucleobase-modifying enzyme and 2) a nucleic acid binding protein that can be programmed to bind to a specific nucleic acid sequence.

[0052] In some embodiments, the nucleobase editor comprises a DNA-binding domain (e.g., a programmable DNA-binding domain such as dCas9 or nCas9) that directs it to a target sequence. In some embodiments, the nucleobase editor comprises a nucleobase-modifying enzyme fused to the programmable DNA-binding domain (e.g., dCas9 or nCas9). A "nucleobase-modifying enzyme" is an enzyme that modifies a nucleobase, converting one nucleobase to another (e.g., a deaminase such as cytosine deaminase or adenosine deaminase). In some embodiments, the nucleobase editor can target cytosine (C) bases in a nucleic acid sequence and convert the C to a thymine (T) base. In some embodiments, the C to T editing is performed by a deaminase, e.g., cytosine deaminase. Base editors capable of other types of base conversions (e.g., adenosine (A) to guanine (G), C to G) are also contemplated.

[0053] In some embodiments, the nucleobase editor that converts C to T comprises a cytosine deaminase. "Cytosine deaminase" refers to an enzyme that catalyzes the chemical reaction "cytosine + HO → uracil + NH" or "5-methyl-cytosine + HO → thymine + NH." As may be apparent from the reaction equation, such a chemical reaction results in a nucleobase change from C to U / T. With respect to a gene, such a nucleotide change or mutation can then result in an amino acid change in the protein, which may affect the function of the protein, for example, a loss of function or a gain of function. In some embodiments, the C to T nucleobase editor comprises dCas9 or nCas9 fused to a cytosine deaminase. In some embodiments, the cytosine deaminase domain is fused to the N-terminus of dCas9 or nCas9. In some embodiments, the nucleobase editor further comprises a domain that inhibits uracil glycosylase and / or a nuclear localization signal.Such nucleobase editors have been described in the art, for example, in U.S. Patent 9,068,179, U.S. Patent Application Publications US 2015 / 0166980 published June 18, 2015; US 2015 / 0166981 published June 18, 2015; US 2015 / 0166982 published June 18, 2015; US 2015 / 0166984 published June 18, 2015; and US 2015 / 0165054 published June 18, 2015; and U.S. Provisional Application, USSN 62 / 245,828 filed October 23, 2015; USSN 62 / 279,346 filed July 15, 2016; and USSN 62 / 279,346 filed March 22, 2016. 62 / 311,763; USSN 62 / 322,178, filed April 13, 2016; USSN 62 / 357,352, filed June 30, 2016; USSN 62,370,700, filed August 3, 2016; USSN 62 / 398,490, filed September 22, 2016; and USSN 62 / 408,686, filed October 14, 2016; PCT application PCT / US2016 / 058344, filed October 22, 2016; U.S. patent application, USSN 15 / 311,852, filed October 22, 2016; and Komor et al., Nature, Programmable editing of target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), the entire contents of each of which are incorporated herein by reference.

[0054] In some embodiments, the nucleobase editor converts A to G. In some embodiments, the nucleobase editor comprises an adenosine deaminase. "Adenosine deaminase" is an enzyme involved in purine metabolism. It is required for the degradation of adenosine from food and for the turnover of nucleic acids in tissues. Its primary function in humans is the development and maintenance of the immune system. With respect to DNA, adenosine deaminase catalyzes the deamination of adenosine by hydrolysis (forming inosine, which base pairs as G). There are no known adenosine deaminases that act on DNA. Instead, known adenosine deaminase enzymes act only on RNA (tRNA or mRNA). Evolved deoxyadenosine deaminase enzymes that accept DNA substrates and deaminate dA to deoxyinosine, and their use in adenosine nucleobase editors herein, are described, for example, in U.S. Provisional Application No. USSN 62 / 370,684, filed August 3, 2016; U.S. Provisional Application No. USSN 62 / 370,684, filed February 3, 2017; U.S. Provisional Application No. USSN 62 / 473,714, filed March 20, 2017; and PCT application PCT / US2017 / 045381, filed August 3, 2017, each of which is incorporated by reference herein. A non-limiting example of an evolved adenosine deaminase that accepts DNA as a substrate is provided in Example 1.

[0055] In some embodiments, the adenosine deaminase is E. coli TadA (SEQ ID NO: 314). Possible mutations in constructs expressing nucleobase editors comprising ecTadA and modified ecTadA are provided in Table 2. Sequences of exemplary EcTadA mutants and nucleobase editors comprising such mutants are provided in Example 1. Table 2. EcTadA mutants for A to G nucleobase editors [Table 1-1]

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Table 1-14

Table 1-15

[0056] In some embodiments, the A to G nucleobase editor comprises dCas9 or nCas9 fused to an adenosine deaminase. Such nucleobase editors are described in U.S. Provisional Application No. USSN 62 / 370,684, filed August 3, 2016; U.S. Provisional Application No. USSN 62 / 370,684, filed February 3, 2017; U.S. Provisional Application No. USSN 62 / 473,714, filed March 20, 2017; and PCT Application No. PCT / US2017 / 045381, filed August 3, 2017, each of which is incorporated herein by reference.

[0057] In some embodiments, the A to G nucleobase editor comprises the structure NH2-[second adenosine deaminase]-[first adenosine deaminase]-[dCas9]-COOH. In some embodiments, the second adenosine deaminase is wild-type ecTadA (SEQ ID NO: 314). In some embodiments, a linker is used between each domain. In some embodiments, the linker is 32 amino acids in length and comprises the amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 384).

[0058] In some embodiments, the adenosine deaminase comprises one or more of the W23X, H36X, N37X, P48X, I49X, R51X, N72X, L84X, S97X, A106X, D108X, H123X, G125X, A142X, S146X, D147X, R152X, E155X, I156X, K157X, and / or K161X mutations in SEQ ID NO: 314, or one or more corresponding mutations in another adenosine deaminase, wherein the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the W23L, W23R, H36L, P48S, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and / or K157N mutations in SEQ ID NO: 314, or one or more corresponding mutations in another adenosine deaminase.

[0059] In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N mutations in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.

[0060] In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations selected from H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 mutations selected from H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of the H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F and K157N mutations in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.

[0061] In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, E155X, I156X, and K157X in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of the W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F and K157N mutations in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.

[0062] In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, A142X, S146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations selected from W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of the W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, R152P, E155V, I156F and K157N mutations in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.

[0063] In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mutations selected from W23X, H36X, P48X, R51X, L84X, A106X, D108X, H123X, S146X, D147X, R152X, E155X, I156X, and K157X in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mutations selected from W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises or consists of the W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F and K157N mutations in SEQ ID NO: 314, or corresponding mutations in another adenosine deaminase.

[0064] In some embodiments, the nucleobase editor converts C to G. Such nucleobase editors are described in U.S. Provisional Application No. USSN 62 / 470, filed March 10, 2017, and U.S. Provisional Application No. USSN 62 / 470,175, filed March 10, 2017, which are incorporated herein by reference.

[0065] Non-limiting, exemplary types of nucleobase editors (including C to T, A to G, and C to G nucleobase editors) and their respective sequences are provided in Example 1. In some embodiments, a nucleobase editor is a variant of a nucleobase editor described herein. For example, in some embodiments, a nucleobase editor is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a nucleobase editor described herein (exemplary sequences are provided in Example 1). In some embodiments, a nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than any of the nucleobase editors provided herein. In some embodiments, a nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 500 amino acids or less, 450 amino acids or less, 400 amino acids or less, 350 amino acids or less, 300 amino acids or less, 250 amino acids or less, 200 amino acids or less, 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids longer or shorter) than any of the nucleobase editors provided herein.

[0066] "Deaminase" refers to an enzyme that catalyzes the removal or deamination of amine groups from molecules, for example, through hydrolysis. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the deamination of cytidine (C) to uridine (U), deoxycytidine (dC) to deoxyuridine (dU), or 5-methyl-cytidine to thymidine (T, 5-methyl-U), respectively. Subsequent DNA repair mechanisms ensure that dU is replaced by T, as described in Komor et al. (Nature, Programmable editing of target base in genomic DNA without double-stranded DNA cleavage, 533, 420-424 (2016), incorporated herein by reference). In some embodiments, the deaminase is a cytosine deaminase, which catalyzes and promotes the conversion of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA). In some embodiments, the deaminase is an adenosine deaminase that converts A to G. In some embodiments, the deaminase is a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is a variant of a naturally occurring deaminase from an organism, wherein the variant does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from an organism. In some embodiments, the deaminase comprises an amino acid sequence that is shorter or longer in length than any of the deaminases provided herein (e.g., no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1%, longer or shorter).In some embodiments, the deaminase comprises an amino acid sequence that is shorter or longer in length (e.g., 100 amino acids or less, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, 10 amino acids or less, 5 amino acids or less, 2 amino acids or less, longer or shorter) than any of the deaminases provided herein.

[0067] A "split nucleobase editor" refers to a nucleobase editor that is provided as an N-terminal portion (also referred to as the N-terminal half) and a C-terminal portion (also referred to as the C-terminal half), encoded by two separate nucleic acids. Polypeptides corresponding to the N-terminal and C-terminal portions of a nucleobase editor can be combined to form a complete nucleobase editor. In some embodiments, for nucleobase editors comprising dCas9 or nCas9, the "split" is located in the dCas9 or nCas9 domain at a site described herein in the split Cas9. Thus, in some embodiments, the N-terminal portion of the nucleobase editor comprises the N-terminal portion of the split Cas9, and the C-terminal portion of the nucleobase editor comprises the C-terminal portion of the split Cas9. Similarly, an intein-N or intein-C can be fused to the N-terminal portion or the C-terminal portion of a nucleobase editor, respectively, to link the N-terminal and C-terminal portions of the nucleobase editor to form a complete nucleobase editor.

[0068] Two proteins or protein domains are considered to be "fused" when a peptide bond is formed between them. In some embodiments, a linker (e.g., a peptide linker) is present between the two proteins or two protein domains. The term "linker" as used herein refers to a chemical group or molecule that links two molecules or moieties, such as two domains of a fusion protein (e.g., a nuclease-inactivating Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain)). Typically, a linker is located between or adjacent to two groups, molecules, or other moieties, and is linked to each other via a covalent bond, thereby linking the two. In some embodiments, the linker is one amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 100 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0069] "Uracil glycosylase inhibitor (UGI)" refers to a protein that inhibits the activity of uracil-DNA glycosylase. Suitable UGI proteins for use in accordance with the present disclosure include, for example, those published in Wang et al., J. Biol. Chem. 264:1163-1171 (1989); Lundquist et al., J. Biol. Chem. 272:21408-21419 (1997); Ravishankar et al., Nucleic Acids Res. 26:4880-4887 (1998); and Putnam et al., J. Mol. Biol. 287:331-346 (1999), each of which is incorporated herein by reference. Non-limiting exemplary proteins that can be used as IUGs of the present disclosure, and their respective sequences, are provided in Example 1. In some embodiments, UGI is a variant of naturally occurring deaminase from an organism, and the variant does not exist in nature.For example, in some embodiments, UGI is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to naturally occurring UGI from an organism or any UGI provided herein (e.g., in Example 1).In some embodiments, UGI comprises an amino acid sequence that is shorter or longer (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than any UGI provided herein. In some embodiments, the UGI comprises an amino acid sequence that is shorter or longer in length (e.g., 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 5 amino acids or less, 2 amino acids longer or shorter) than any UGI provided herein.

[0070] gRNA is a component of the CRISPR / Cas system. The term "gRNA" (guide ribonucleic acid) refers herein to the fusion of a CRISPR-targeting RNA (crRNA) and a trans-activating crRNA (tracrRNA), which provides both targeting specificity and scaffolding / binding capabilities for the Cas9 nuclease. A "crRNA" is a bacterial RNA that confers targeting specificity and requires a tracrRNA to bind to Cas9. A "tracrRNA" is a bacterial RNA that links the crRNA to the Cas9 nuclease and can typically bind to any crRNA. The sequence specificity of the Cas DNA-binding protein is determined by the gRNA, which has complementary nucleotide base pairing to the target DNA sequence. Native gRNAs contain a 20-nucleotide (nt) specificity-determining sequence (SDS) that specifies the DNA sequence to be targeted, followed immediately by an 80-nt scaffold sequence, which allows the gRNA to bind to Cas9. In some embodiments, the SDS of the present disclosure has a length of 15 to 100 nucleotides or more. For example, the SDS may have a length of 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, or 15-20 nucleotides. In some embodiments, the SDS is 20 nucleotides long. For example, the SDS may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. At least a portion of the target DNA sequence is complementary to the SDS of the gRNA. For Cas9 to successfully bind to a DNA target sequence, a region of the target sequence must be complementary to the SDS of the gRNA sequence, immediately followed by the correct protospacer adjacent motif (PAM) sequence (e.g., NGG for Cas9 and TTN, TTTN, or YTN for Cpf1). In some embodiments, an SDS is 100% complementary to its target sequence. In some embodiments, an SDS sequence has less than 100% complementarity to its target sequence and is therefore considered to be partially complementary to the target sequence.For example, the targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence. In some embodiments, the SDS of the template DNA or target DNA may differ from the complementary region of the gRNA by 1, 2, 3, 4, or 5 nucleotides.

[0071] In addition to the SDS, the gRNA contains a scaffold sequence (corresponding to the tracrRNA in the native CRISPR / Cas system) required for its binding to Cas9 (referred to herein as the "gRNA handle"). In some embodiments, the gRNA comprises the structure 5'-[SDS]-[gRNA handle]-3'. In some embodiments, the scaffold sequence comprises the nucleotide sequence 5'-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3' (SEQ ID NO: 358). Other non-limiting, suitable gRNA handle sequences that can be used in accordance with the present disclosure are listed in Table 1. Table 1. Guide RNA handle sequences [Table 2-1] [Table 2-2]

[0072] In some embodiments, the guide RNA is about 15-120 nucleotides in length and comprises a sequence of at least 10 contiguous nucleotides complementary to the target sequence. , 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides in length. In some embodiments, the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous nucleotides complementary to the target sequence. Sequence complementarity refers to the distinguishable interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine.

[0073] A "protospacer adjacent motif" (PAM) is typically a sequence of nucleotides located adjacently (e.g., within 10, 9, 8, 7, 6, 5, 4, 3, 3, or 1 nucleotide of the target sequence). A PAM sequence is "immediately adjacent" to a target sequence if it is adjacent to the target sequence (i.e., there are no nucleotides located between the PAM sequence and the target sequence). In some embodiments, the PAM sequence is a wild-type PAM sequence. Examples of PAM sequences include, but are not limited to, NGG, NGR, NNGRR(T / N), NNNNGATT, NNAGAAW, NGGAG, NAAAC, AWG, and CC. In some embodiments, the PAM sequence is obtained from Streptococcus pyogenes (e.g., NGG or NGR). In some embodiments, the PAM sequence is obtained from Staphylococcus aureus (e.g., NNGRR(T / N)). In some embodiments, the PAM sequence is obtained from Neisseria meningitidis (e.g., NNNNGATT). In some embodiments, the PAM sequence is obtained from Streptococcus thermophilus (e.g., NNAGAAW or NGGAG). In some embodiments, the PAM sequence is obtained from Treponema denticola (e.g., NAAAAC). In some embodiments, the PAM sequence is obtained from Escherichia coli (e.g., AWG). In some embodiments, the PAM sequence is obtained from Pseudomonas auruginosa (e.g., CC). Other PAM sequences are contemplated. The PAM sequence is typically located downstream (i.e., 3') from the target sequence, although in some embodiments, the PAM sequence may be located upstream (i.e., 5') from the target sequence.

[0074] A "nuclear localization signal" or "NLS" refers to an amino acid sequence that "tags" a protein for import into the nucleus of a cell by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. One or more NLSs can be added to the N- or C-terminus of a protein, or internally (e.g., between two protein domains). For example, one or more NLSs can be added to the N- or C-terminus of a nucleobase editor, or between Cas9 and the deaminase in a nucleobase editor. In some embodiments, one, two, three, four, five, or more NLSs can be added. Nuclear localization sequences are known in the art and will be apparent to those skilled in the art. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, filed November 23, 2000, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 373) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 374). In some embodiments, a linker is inserted between the Cas9 and the deaminase.

[0075] NLSs can be classified as monopartite or bipartite. A non-limiting example of a monopartite NLS is the sequence PKKKRKV (SEQ ID NO: 373) in the SV40 large T antigen. A bipartite NLS typically comprises two clusters of basic amino acids separated by a spacer of about 10 amino acids. One non-limiting example of a bipartite NLS is the nucleoplasmin NLS, KRPAATKKAGQAKKKK (spacer underlined) (SEQ ID NO: 344). In some embodiments, an NLS that can be used in accordance with the present disclosure is a nucleoplasmin NLS comprising the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO: 344). Other bisecting NLSs that can be used in accordance with the present disclosure include, without limitation, the SV40 bisecting NLS (KRTADGSEFESPKKKRKV (SEQ ID NO: 375), described, for example, in Hodel et al., J Biol Chem. 2001 Jan 12;276(2):1317-25, incorporated herein by reference); the Kanadaptin bisecting NLS (KKTELQTTNAENKTKKL (SEQ ID NO: 345), described, for example, in Hubner et al., Biochem J. 2002 Jan 15;361(Pt 2):287-96, incorporated herein by reference); the influenza A nucleoprotein bisecting NLS (KRGINDRNFWRGENGRKTR (SEQ ID NO: 346), described, for example, in Ketha et al., BMC Cell Biology, incorporated herein by reference. 2008;9:22); and ZO-2 bisecting NLS (RKSGKIAAIVVKRPRK (SEQ ID NO: 347), described, for example, in Quiros et al., Nusrat A (eds.), Molecular Biology of the Cell. 2013;24(16):2528-2543, incorporated herein by reference).

[0076] A nucleotide sequence encoding an NLS is "operably linked" to a nucleotide sequence encoding a protein (e.g., Cas9 or a nucleobase editor) to which it is fused when the two coding sequences are "in-frame with each other" and translated as a single polypeptide fusing the two sequences.

[0077] Nucleic acids of the present disclosure may contain one or more genetic elements. "Genetic element" refers to a specific nucleotide sequence that plays a role in nucleic acid expression (e.g., promoter, enhancer, terminator) or encodes another product of the engineered nucleic acid (e.g., a nucleotide sequence encoding a guide RNA, a protein, and / or an RNA interference molecule).

[0078] A "promoter" refers to a regulatory region of a nucleic acid sequence at which the rate of initiation and subsequent transcription of the nucleic acid sequence is controlled. A promoter also includes subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Promoters can be constitutive, inducible, activatable, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or drives the transcription of a nucleic acid sequence that it regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates so as to control ("drive") the transcription initiation and / or expression of that sequence.

[0079] A promoter may be one naturally associated with a gene or sequence, such as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter is referred to as an "endogenous promoter." In some embodiments, a coding nucleic acid sequence is placed under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring," such as those containing different elements of different transcriptional regulatory regions and / or mutations that alter expression through genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR).

[0080] In some embodiments, a promoter used in accordance with the present disclosure is an "inducible promoter," which is a promoter characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence, under the influence, or contact of an inducer signal. The inducer signal may be endogenous, or typically an exogenous condition (e.g., light), compound (e.g., a chemical or non-chemical compound), or protein that contacts the inducible promoter in such a way that it is active in controlling transcriptional activity from the inducible promoter. Thus, a "signal that modulates transcription" of a nucleic acid refers to an inducer signal acting on an inducible promoter. A signal that modulates transcription can activate or inactivate transcription, depending on the regulatory system used. Activating transcription can involve directly acting on the promoter to drive transcription or indirectly acting on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, deactivating transcription can involve directly acting on the promoter to prevent transcription or indirectly acting on the promoter by activating a repressor that then acts on the promoter.

[0081] A "transcription terminator" is a nucleic acid sequence that terminates transcription. Transcription terminators can be unidirectional or bidirectional. They consist of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. A transcription terminator sequence prevents an upstream promoter from activating the transcription of a downstream nucleic acid sequence. A transcription terminator may be necessary in vivo to achieve a desired expression level or to prevent transcription of a specific sequence. A transcription terminator is considered "operably linked" to a nucleotide sequence if it is capable of terminating the transcription of the sequence to which it is linked.

[0082] The most commonly used type of terminator is a forward terminator. When placed upstream of a nucleic acid sequence that is normally transcribed, a forward transcription terminator will interrupt transcription. In some embodiments, a bidirectional transcription terminator is provided, which typically stops transcription on both the forward and reverse strands. In some embodiments, a reverse transcription terminator is provided, which typically terminates transcription on the reverse strand only.

[0083] In prokaryotic systems, terminators typically fall into two categories: (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators generally consist of a palindromic sequence that forms a stem-loop rich in GC base pairs followed by several T bases. While not wishing to be bound by theory, the conventional model of transcription termination is that the stem-loop pauses the RNA polymerase, and transcription of the polyA tail unwinds the RNA:DNA duplex, dissociating it from the RNA polymerase.

[0084] In eukaryotic systems, terminator regions may contain specialized DNA sequences that allow for site-specific cleavage of nascent transcripts to expose polyadenylation sites. This signals specialized endogenous polymerases to add a stretch of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are thought to be more stable and more efficiently translated. Thus, in some eukaryotic embodiments, terminators may contain signals for RNA cleavage. In some embodiments, terminator signals promote polyadenylation of the message. Elements of the terminator and / or polyadenylation site can serve to enhance output nucleic acid levels and / or minimize readthrough between nucleic acids.

[0085] Terminators for use according to the present disclosure include any transcription terminator described herein or known to those skilled in the art. Examples of terminators include, but are not limited to, gene termination sequences such as the bovine growth hormone terminator, and viral termination sequences such as the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB T1, hisLGDCBHAFI, metZWV, rrnC, xapR, aspA, and arcA terminators. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as one resulting from sequence truncation.

[0086] The "woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE)" is a DNA sequence that, when transcribed, creates a tertiary structure that enhances expression. It is commonly used in molecular biology to enhance expression of genes delivered by viral vectors. The WPRE is a tripartite regulatory element, having gamma, alpha, and beta components. The full-length WPRE sequence is 609 bp in length: GCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCG (SEQ ID NO: 376).

[0087] "Adeno-associated virus" or "AAV" is a virus that infects humans and some other primate species. The wild-type AAV genome is a single-stranded deoxyribonucleic acid (ssDNA), which may be the sense or antisense strand. The genome contains two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs): rep and cap, located between the ITRs. The rep ORF contains four overlapping genes encoding the Rep proteins required for the AAV life cycle. The cap ORF contains overlapping genes encoding the capsid proteins VP1, VP2, and VP3, which interact together to form the viral capsid. VP1, VP2, and VP3 are translated from a single mRNA transcript that can be spliced ​​in two different ways: longer or shorter introns can be excised, resulting in the formation of two mRNA isoforms: approximately 2.3 kb and approximately 2.6 kb long. The capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a non-enveloped T-1 icosahedral lattice that can protect the AAV genome. The mature capsid consists of VP1, VP2, and VP3 in a ratio of approximately 1:1:10 (molecular weights of approximately 87, 73, and 62 kDa, respectively).

[0088] The rAAV particle may comprise a nucleic acid vector (e.g., a recombinant genome), which may minimally comprise: (a) one or more heterologous nucleic acid regions comprising one or more nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest (e.g., a split Cas9 or split nucleobase) or an RNA of interest (e.g., a gRNA), or a sequence encoding a Rep protein; and (b) one or more regions comprising inverted terminal repeats (ITRs) (e.g., wild-type ITR sequences or engineered ITR sequences) flanked by one or more nucleic acid regions (e.g., heterologous nucleic acid regions). In some embodiments, the nucleic acid vector is 4 kb to 5 kb in size (e.g., 4.2 to 4.7 kb in size). In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein. In some embodiments, the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, the double-stranded nucleic acid vector may be, for example, a self-complementary vector, which includes a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, which initiates the formation of a double-stranded nucleic acid vector.

[0089] The terms "nucleic acid" and "polynucleotide," as used herein, refer to a compound comprising a nucleic acid base and an acidic moiety (e.g., a nucleotide or a polymer of nucleotides). Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably and refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids can be naturally occurring, e.g., in the context of a genome, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. Alternatively, a nucleic acid molecule may be a non-naturally occurring molecule, such as a recombinant DNA or RNA, an artificial chromosome, an engineered genome (e.g., an engineered viral vector), an engineered vector, or fragments thereof, or a synthetic DNA, RNA, or DNA / RNA hybrid, optionally containing non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids may be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, or the like. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids may include nucleoside analogs, such as analogs with chemically modified bases or sugars and backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, C5-methylcytidine, C5-methyl- ... adenosine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0090] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified by the addition of a chemical moiety, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, or a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof. The term "fusion protein," as used herein, refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) end of the protein, thereby forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. The protein may comprise different domains of a nucleic acid editing protein, such as a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9, which directs binding of the protein to a target site), and a nucleic acid cleavage domain or catalytic domain. In some embodiments, the protein is in a complex with or associated with a nucleic acid, such as RNA or DNA. Any of the proteins provided herein can be produced by any method known in the art.For example, the proteins provided herein can be produced through recombinant protein expression and purification, which is particularly suitable for fusion proteins that contain peptide linkers.Methods for recombinant protein expression and purification are well known, including those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which is incorporated herein by reference.

[0091] The term "subject," as used herein, refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent (e.g., a mouse, a rat). In some embodiments, the subject is a domesticated animal. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either gender and at any stage of development.

[0092] The term "recombinant," as used herein with respect to proteins or nucleic acids, refers to proteins that do not occur in nature but are the product of artificial manipulation. For example, in some embodiments, recombinant proteins or nucleic acid molecules comprise an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence. The fusion proteins (e.g., base editors) described herein are produced by recombinant means. Recombinant techniques are known to those skilled in the art.

[0093] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent that encapsulates a material, that is involved in carrying or transporting a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not toxic to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.).

[0094] As used herein, a "therapeutically effective amount" refers to the amount of each therapeutic agent (e.g., nucleobase editor, rAAV) described in this disclosure, alone or in combination with one or more other therapeutic agents, required to confer a therapeutic effect on a subject. As will be recognized by those skilled in the art, effective amounts will vary depending on the particular condition being treated, the severity of the condition, individual subject parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of a healthcare professional. These factors are well known to those skilled in the art and can be addressed using only routine experimentation. It is generally preferred to use maximum doses of the individual components or combinations thereof, i.e., the highest safe dose according to sound medical judgment. As will be understood by those skilled in the art, subjects may insist on lower or tolerable doses for medical, psychological, or virtually any other reason. Empirical considerations such as half-life will generally contribute to determining dosages. For example, therapeutic agents compatible with the human immune system, such as humanized antibodies or polypeptides containing regions from fully human antibodies, may be used to extend the half-life of the polypeptide and to prevent the polypeptide from being attacked by the host's immune system.

[0095] A "subject in need thereof" refers to an individual having a disease, signs and / or symptoms of a disease, or predisposition to a disease, and the objective is to cure, alleviate, relieve, alter, treat, ameliorate, improve, or affect the disease, symptoms of a disease, or predisposition to a disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. In some embodiments, the mammal is a rodent. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat. In some embodiments, the mammal is a companion animal. "Companion animal" refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; farm animals such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. The terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., taking into account a history of symptoms and / or taking into account genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0096] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Provided herein are compositions (e.g., vectors, recombinant viruses) and kits comprising nucleic acids encoding split Cas9 proteins or nucleobase editors, as well as methods for using such nucleic acids to deliver nucleobase editors or Cas9 proteins into cells. The N-terminal and C-terminal portions of the nucleobase editor or Cas9 protein are encoded on separate nucleic acids and delivered into cells, for example, via recombinant adeno-associated virus (rAAV particle) delivery. Polypeptides corresponding to the N-terminal and C-terminal portions of a nucleobase editor or Cas9 protein may be linked to form the complete nucleobase editor or Cas9 protein, e.g., through intein-mediated protein splicing.

[0097] Accordingly, some aspects of the present disclosure relate to compositions comprising: (i) a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein, wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal (NLS).

[0098] The Cas9 protein encoded by the first and second nucleotide sequences is referred to herein as "split Cas9." Cas9 proteins are known to have an N-terminal lobe and a C-terminal lobe connected by an unusual linker (e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, which is incorporated herein by reference). In some embodiments, the N-terminal portion of the split Cas9 protein comprises the N-terminal lobe of the Cas9 protein. In some embodiments, the C-terminal portion of the split Cas9 protein comprises the C-terminal lobe of the Cas9 protein. In some embodiments, the N-terminal portion of the split Cas9 comprises a portion of any one of SEQ ID NOS: 1-275 and 394-397, which corresponds to amino acids 1-(550-650) of SEQ ID NOS: 1. "1-(550-650)" means editing starting at amino acid 1 and anywhere between amino acids 550 and 650 (inclusive).For example, the N-terminal portion of split Cas9 may be composed of amino acids 1 to 550, 1 to 551, 1 to 552, 1 to 553, 1 to 554, 1 to 555, 1 to 556, 1 to 557, 1 to 558, 1 to 559, 1 to 560, 1 to 561, 1 to 562, 1 to 563, 1 to 564, 1 to 565, 1 to 566, 1 to 567, 1 to 568, 1 to 569, 1 to 570, 1 to 571, 1 to 572, 1 to 573, 1 to 574, 1 to 575, 1 to 576, 1 to 577, 1 to 578, 1 to 579, 1 to 580, 1 to 581, 1 to 582, 1 to 583, 1 to 584, 1 to 585, 1 to 586, 1 to 587, 1 to 588, 1 to 589, 1 to 590, 1 to 591, 1 to 592, 1 to 593, 1 to 594, 1 to 595, 1 to 596, 1 ​​to 597, 1 to 598, 1 to 599, 1 to 590, 1 to 591, 1 to 599, 1 to 599, 1 to 590, 1 to 591, 1 to 592, 1 to 593, 1 to 594, 1 to 595, 1 to 596, 1 ​​to 5 , 1~573, 1~574, 1~575, 1~576, 1~577, 1~578, 1~579, 1~580, 1~581, 1~582, 1~583, 1~584, 1~585, 1~586, 1~587, 1~588, 1~589, 1~590, 1~591, 1~592, 1~593, 1~594, 1~595, 1~596, 1~597, 1~598, 1~599, 1~600, 1~601 01, 1~602, 1~603, 1~604, 1~605, 1~606, 1~607, 1~608, 1~609, 1~610, 1~611, 1~612, 1~613, 1~614, 1~615, 1~616, 1~617, 1~618, 1~619, 1~620, 1~621, 1~622, 1~623, 1~624, 1~625, 1~626, 1~627, 1~628, 1~629, 1 In some embodiments, the N-terminal portion of the split Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397 corresponding to amino acids 1-573 or 1-637 of SEQ ID NO: 1.

[0099] The C-terminal portion of a split Cas9 can be linked to the N-terminal portion of a split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein begins where the N-terminal portion of the Cas9 protein ends. Thus, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, which corresponds to amino acids (551-651)-1368 of SEQ ID NO: 1. "(551-651)-1368" refers to a start at amino acids between amino acids 551 and 651 (inclusive) and an edit at amino acid 1368.For example, the C-terminal portion of split Cas9 may be selected from amino acids 551 to 1368, 552 to 1368, 553 to 1368, 554 to 1368, 555 to 1368, 556 to 1368, 557 to 1368, 558 to 1368, 559 to 1368, 560 to 1368, 561 to 1368, 562 to 1368, 563 to 1368, 564 to 1368, 565 to 1368, 566 to 1368, 567 to 1368, 568 to 1368, 569 to 1368, 570 to 1368, 571 to 1368, 572 to 1368, 573 to 1368, and 574 to 1368 of SEQ ID NO: 1. , 575~1368, 576~1368, 577~1368, 578~1368, 579~1368, 580~1368, 581~1368, 582~1368, 583~1368, 584~1368, 585~1368, 586~1368, 587~1368, 588~1 368, 589~1368, 590~1368, 591~1368, 592~1368, 593~1368, 594~1368, 595~1368, 596~1368, 597~1368, 598~1368, 599~1368, 600~1368, 601~1368, 602 ~1368, 603~1368, 604~1368, 605~1368, 606~1368, 607~1368, 608~1368, 609~1368, 610~1368, 611~1368, 612~1368, 613~1368, 614~1368, 615~1368, 616~1368, 617~1368, 618~1368, 619~1368, 620~1368, 621~1368, 622~1368, 623~1368, 624~1368, 625~1368, 626~1368, 627~1368, 628~1368, 629~136 8, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368.In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, corresponding to amino acids 574-1368 or 638-1368 of SEQ ID NO: 1.

[0100] Cas9 variants may also be delivered to cells using the methods described herein. For example, the Cas9 variant may be a "split" variant as described herein. The Cas9 variant may comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 sequences provided herein. In some embodiments, the Cas9 variant comprises an amino acid sequence that is shorter or longer (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than any of the Cas9 proteins provided herein (e.g., those in Example 1). In some embodiments, the UGI comprises an amino acid sequence that is shorter or longer in length than any of the Cas9 proteins provided herein (e.g., 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids or less, or 2 amino acids or less, longer or shorter).

[0101] In some embodiments, the N-terminal portion of the split Cas9 comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding portion of any one of the Cas9 sequences provided herein (e.g., in Example 1). In some embodiments, the N-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein. In some embodiments, the N-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g., 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids or less, or 2 amino acids or less, longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein.

[0102] In some embodiments, the C-terminal portion of the split Cas9 comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding portion of any one of the Cas9 sequences provided herein (e.g., in Example 1). In some embodiments, the C-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein. In some embodiments, the C-terminal portion of the split Cas9 comprises an amino acid sequence that is shorter or longer in length (e.g., 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids or less, or 2 amino acids or less, longer or shorter) than the corresponding portion of any of the Cas9 proteins provided herein.

[0103] In some embodiments, the Cas9 variant is dCas9 or nCas9. In some embodiments, the N-terminal portion of the split Cas9 comprises a mutation corresponding to the D10A mutation in SEQ ID NO: 1. In some embodiments, the N-terminal portion of the split Cas9 comprises a mutation corresponding to the D10A mutation in SEQ ID NO: 1, and the C-terminal portion of the split Cas9 comprises a mutation corresponding to the H840A mutation in SEQ ID NO: 1. In some embodiments, the N-terminal portion of the split Cas9 comprises a mutation corresponding to the D10A mutation in SEQ ID NO: 1, and the C-terminal portion of the split Cas9 comprises a histidine at a position corresponding to position 840 in SEQ ID NO: 1.

[0104] In some embodiments, an intein system may be used to link the N-terminal portion of the Cas9 protein to the C-terminal portion of the Cas9 protein. In some embodiments, the N-terminal portion of Cas9 is fused to intein-N. In some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of Cas9 to form the structure NH2-[N-terminal portion of Cas9]-[intein-N]-COOH. In some embodiments, intein-N is encoded by the dnaE-n gene. In some embodiments, intein-N comprises the amino acid sequence of any one of SEQ ID NOs: 350-351 and 354-355. In some embodiments, the C-terminal portion of Cas9 is fused to intein-C, and intein-C is fused to the N-terminus of the C-terminal portion of Cas9 to form the structure NH2-[intein-C]-[C-terminal portion of Cas9]-COOH. In some embodiments, intein-C is encoded by the dnaE-c gene. In some embodiments, Intein-C comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 356-357. Other split intein systems can also be used in the present disclosure and are known in the art.

[0105] Split nucleobase editors may be used in the present disclosure. Some aspects of the present disclosure relate to compositions comprising: (i) a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the nucleobase editor.

[0106] Variants of nucleobase editors are contemplated. For example, variants of nucleobase editors may also be "split" as described herein. Variants of nucleobase editors may comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the nucleobase editor sequences (SEQ ID NO:XX) provided herein.

[0107] In some embodiments, the N-terminal portion of a split nucleobase editor comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding portion of any one of the nucleobase editors provided herein (e.g., in Example 1). In some embodiments, the N-terminal portion of a split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than the corresponding portion of any one of the nucleobase editors provided herein. In some embodiments, the N-terminal portion of a split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids or less, or 2 amino acids or less, longer or shorter) than the corresponding portion of any of the nucleobase editors provided herein.

[0108] In some embodiments, the C-terminal portion of a split nucleobase editor comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding portion of any one of the nucleobase editors provided herein (e.g., in Example 1). In some embodiments, the C-terminal portion of a split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, longer or shorter) than the corresponding portion of any one of the nucleobase editors provided herein. In some embodiments, the C-terminal portion of a split nucleobase editor comprises an amino acid sequence that is shorter or longer in length (e.g., 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 10 amino acids or less, 5 amino acids or less, or 2 amino acids or less, longer or shorter) than the corresponding portion of any of the nucleobase editors provided herein.

[0109] As described herein, the N-terminal portion of the nucleobase editor comprises the N-terminal portion of a nuclease-inactivated Cas9 protein (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the N-terminal portion of the nucleobase editor further comprises a nucleobase-modifying enzyme (e.g., a nuclease, a nickase, a recombinase, a deaminase, a DNA repair enzyme, a DNA-damaging enzyme, a dismutase, an alkylating enzyme, a depurinase, an oxidase, a pyrimidine dimer-forming enzyme, an integrase, a transposase, a polymerase, a ligase, a helicase, a photolyase, a glycosylase, a methylase, an acetylase, a methyltransferase, a demethylase, or an epigenetic modifier, etc.). In some embodiments, the nucleobase-modifying enzyme is a deaminase (e.g., a cytosine deaminase or an adenosine deaminase, or a functional variant thereof). In some embodiments, the nucleobase-modifying enzyme is fused to the N-terminus of the N-terminal portion of a split dCas9 or split nCas9. In some embodiments, the N-terminal portion of the nucleobase editor has the following structure: NH2-[nucleobase-modifying enzyme]-[N-terminal portion of dCas9 or nCas9]-COOH. In some embodiments, the N-terminal portion of the nucleobase editor is fused to an intein-N. In some embodiments, the intein-N is fused to the C-terminus of the N-terminal portion of the nucleobase editor.

[0110] In some embodiments, the first nucleotide sequence encodes a polypeptide comprising the structure NH2-[nucleobase-modifying enzyme]-[N-terminal portion of dCas9 or nCas9]-[intein-N]-COOH.

[0111] In some embodiments, the C-terminal portion of the nucleobase editor comprises the C-terminal portion of a nuclease-inactivated Cas9 protein (dCas9) or Cas9 nickase (nCas9). In some embodiments, a nucleobase-modifying enzyme is fused to the C-terminus of the C-terminal portion of a split dCas9 or split nCas9. In some embodiments, the C-terminal portion of the nucleobase editor is of the structure: NH2-[C-terminal portion of dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH. In some embodiments, the C-terminal portion of the nucleobase editor comprises an intein-C fused to the C-terminal portion of a Cas9 protein. In some embodiments, the intein-C is fused to the N-terminus of the C-terminal portion of the nucleobase editor. In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH2-[intein-C]-[C-terminal portion of a Cas9 protein]-COOH.

[0112] In some embodiments, the N-terminal portion of the split nucleobase editor further comprises an inhibitor of uracil glycosylase (UGI). In some embodiments, the first nucleotide sequence encodes a polypeptide of the structure: NH2-[UGI]-[nucleobase-modifying enzyme]-[N-terminal portion of dCas9 or nCas9]-[intein-N]. In some embodiments, the first nucleotide sequence encodes a polypeptide of the structure: NH2-[nucleobase-modifying enzyme]-[UGI]-[N-terminal portion of dCas9 or nCas9]-[intein-N].

[0113] In some embodiments, the C-terminal portion of the split nucleobase editor further comprises an enzyme that inhibits the activity of uracil glycosylase (UGI). In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH2-[intein-C]-[C-terminal portion of dCas9 or nCas9]-[UGI]-COOH. In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH2-[intein-C]-[C-terminal portion of dCas9 or nCas9]-[nucleobase-modifying enzyme]-[UGI]-COOH. In some embodiments, the second nucleotide sequence encodes a polypeptide of the structure: NH2-[intein-C]-[C-terminal portion of dCas9 or nCas9]-[UGI]-[nucleobase-modifying enzyme]-COOH.

[0114] In some embodiments, the N-terminal and C-terminal portions of the nucleobase are linked to form a complete split nucleobase editor. In some embodiments, a split nucleobase editor may comprise any one of the following structures: NH2-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-[UGI]-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-[UGI]-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-[UGI]-COOH NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOHNH 2-[UGI]-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-[UGI]-[nucleobase-modifying enzyme]-COOH or NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-[UGI]-COOH.

[0115] In some embodiments, the first nucleotide sequence or the second nucleotide sequence (encoding either a split Cas9 protein or a split nucleobase editor) is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal (NLS). For example, the first nucleotide sequence may be operably linked to a nucleotide sequence encoding one or more (e.g., 2, 3, 4, 5, or more) bipartite NLSs. In some embodiments, the second nucleotide sequence may be operably linked to a nucleotide sequence encoding one or more (e.g., 2, 3, 4, 5, or more) bipartite NLSs. Thus, a split Cas9 or split nucleobase editor formed by linking an N-terminal portion and a C-terminal portion may comprise one or more bipartite NLSs. For example, a split Cas9 or split nucleobase editor may comprise any one of the following structures (bNLS means one or more bipartite nuclear localization signals): NH2-bNLS-[Cas9]-COOH NH2-[Cas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-COOHNH 2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[UGI]-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-[UGI]-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOH NH2-[UGI]-[nucleobase-modifying enzyme]-bNLS[dCas9 or nCas9]-COOH NH2-[UGI]-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-COOHNH 2-bNLS-[UGI]-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-COOH NH2-bNLS-[UGI]-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-COOH NH2-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-COOH NH2-[UGI]-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-COOH NH2-[UGI]-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-COOH NH2-bNLS-[UGI]-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[UGI]-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[UGI]-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-[dCas9 or nCas9]-COOHNH 2-bNLS-[nucleobase-modifying enzyme]-[UGI]-bNLS-[dCas9 or nCas9]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[UGI]-[dCas9 or nCas9]-bNLS-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[UGI]-[dCas9 or nCas9]-bNLS-COOH NH2-[nucleobase-modifying enzyme]-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-[dCas9 or nCas9]-bNLS-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-[UGI]-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-[UGI]-COOH NH2-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-[UGI]-COOH NH2-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-[UGI]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-[UGI]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-[UGI]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-[UGI]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-[UGI]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-COOH NH2-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-COOH NH2-bNLS-[nucleobase-modifying enzyme]-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[UGI]-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH NH2-[UGI]-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH NH2-[UGI]-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-[UGI]-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[UGI]-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[UGI]-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-[UGI]-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[UGI]-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[UGI]-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[UGI]-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-[UGI]-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-bNLS-[UGI]-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-[UGI]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-[UGI]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS[UGI]-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[dCas9 or nCas9]-[UGI]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[dCas9 or nCas9]-[UGI]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-[dCas9 or nCas9]-bNLS-[UGI]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[dCas9 or nCas9]-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[UGI]-bNLS-[nucleobase-modifying enzyme]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-[UGI]-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-[UGI]-COOH NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-[UGI]-COOH NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-[UGI]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-[UGI]-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-[UGI]-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-[UGI]-bNLS-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-[UGI]-bNLS-COOH NH2-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-COOH NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-[UGI]-bNLS-COOH NH2-bNLS-[dCas9 or nCas9]-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-COOH NH2-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-COOH or NH2-bNLS-[dCas9 or nCas9]-bNLS-[nucleobase-modifying enzyme]-bNLS-[UGI]-bNLS-COOH

[0116] As used herein, "NH2-" represents the N-terminus of a protein or polypeptide, and "-COOH" represents the C-terminus of a protein or polypeptide. "]-[" represents a peptide bond or linker. In some embodiments, a linker can be used to link any of the proteins or protein domains described herein. The linker can be simple, such as a covalent bond, or a polymeric linker spanning many atoms in length. In some embodiments, the linker is a polypeptide or based on amino acids. In some embodiments, the linker is not peptide-like. In some embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, a disulfide bond, a carbon-heteroatom bond, etc.). In some embodiments, the linker is a carbon-nitrogen bond of an amide bond. In some embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched, aliphatic or heteroaliphatic linker. In some embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In some embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In some embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In some embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In some embodiments, the linker comprises a polyethylene glycol moiety (PEG). In some embodiments, the linker comprises an amino acid. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises an aryl or heteroaryl moiety. In some embodiments, the linker is based on a phenyl ring. The linker may comprise a functionalized moiety to facilitate the addition of a nucleophile (e.g., thiol, amino) from the peptide to the linker.Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0117] In some embodiments, the linker is a single amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 377), which may also be referred to as an XTEN linker. In some embodiments, the linker comprises the amino acid sequence: SGGS (SEQ ID NO: 378). In some embodiments, the linker comprises the amino acid sequence: (SGGS) n (SEQ ID NO: 379), (GGGS) n (SEQ ID NO: 380), (GGGGS) n (SEQ ID NO: 381), (G) n (SEQ ID NO: 390), (EAAAK) n (SEQ ID NO: 382), (GGS) n , SGSETPGTSESATPES (SEQ ID NO: 377), or (XP) nmotif, or any combination thereof, wherein n is independently an integer from 1 to 30 (inclusive), and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises the amino acid sequence: SGSETPGTSESATPES (SEQ ID NO: 377), and SGGS (SEQ ID NO: 378). In some embodiments, the linker comprises the amino acid sequence: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 383). In some embodiments, the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 384). In some embodiments, the linker comprises the amino acid sequence: GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 385).

[0118] In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 343). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 391). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 392). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 393).

[0119] In some embodiments, the first and second nucleotide sequences are on the same nucleic acid vector. In some embodiments, the first and second nucleotide sequences are on different nucleic acid vectors. In some embodiments, the vector is a plasmid. In some embodiments, the nucleic acid vector is a recombinant genome of an adeno-associated virus (rAAV). In some embodiments, the nucleic acid vector is a genome of an adeno-associated virus packaged in an rAAV particle. In some embodiments, the first and / or second nucleotide sequence is operably linked to a promoter. In some embodiments, the nucleic acid vector further comprises a nucleotide sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs operably linked to the promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

[0120] Inducible promoters of the present disclosure can be induced (or repressed) by one or more physiological conditions, such as changes in light, pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more exogenous or endogenous inducers. Exogenous inducer signals or inducers may include, without limitation, amino acids and amino acid analogs, sugars and polysaccharides, nucleic acids, protein transcription activators and repressors, cytokines, toxins, petroleum-based compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones, or combinations thereof.

[0121] Inducible promoters of the present disclosure include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., rat glucocorticoid receptor, human estrogen receptor, etc.), and the like. These include promoters based on the ecdysone receptor of moths, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse, and human), pathogenicity-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells). Other inducible promoter systems are known in the art and can be used in accordance with the present disclosure.

[0122] In some embodiments, the inducible promoter of the present disclosure functions in prokaryotic cells (e.g., bacterial cells). Examples of inducible promoters for use in prokaryotic cells include, but are not limited to, bacteriophage promoters (e.g., Pls1con, T3, T7, SP6, PL), and bacterial promoters (e.g., Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, Pm), or hybrids thereof (e.g., PLlacO, PLtetO).Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters, such as the positively regulated σ70 promoter (e.g., inducible pBad / araC promoter, right promoter of the Lux cassette, modified lambda Prm promoter, plac Or2-62(positive), pBad / AraC with extra REN sites, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), σS promoters (e.g., Pdps), σS32 promoters (e.g., heat shock), and σS54 promoters (e.g., glnAp2); negatively regulated E. coli promoters, such as the negatively regulated σ70 promoter (e.g., promoter(PRM+), modified lambda Prm promoter, TetR-TetR-4C P(Las)TetO, P(Las)CIO, P(Lac)IQ, RecA_DlexO_DLacO1, dapAp, FecA, Pspac-hy, pcI, plux-cI, plux-lac, CinR, CinL, glucose-regulated modified Pr, modified Prm+, FecA, Pcya, rec A(SOS), Rec A(SOS), EmrR_regulated, BetI_regulated, pLac_lux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cI, pLux / cI, LacI, LacIQ, pLacIQ1, pLas / cI, pLas / Lux, pLux / Las, pRecA with LexA binding site, reverse BBa_R0011, pLacI / ara-1, pLacIq, rrnB P1, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), σ promoters (e.g., Lutz-Bujard LacO with alternative σ factor σ), σ promoters (e.g., Lutz-Bujard LacO with alternative σ factor σ), and σ promoters (e.g., glnAp2); negatively regulated B. subtilis promoters, e.g., repressible B. subtilis σ promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σ promoters.Other inducible microbial promoters can be used in accordance with the present disclosure.

[0123] In some embodiments, the inducible promoter of the present disclosure functions in eukaryotic cells (e.g., mammalian cells). Examples of inducible promoters for use in eukaryotic cells include, but are not limited to, chemically regulated promoters (e.g., alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, and pathogenicity-related (PR) promoters) and physiologically regulated promoters (e.g., temperature-regulated promoters and light-regulated promoters).

[0124] Recombinant adeno-associated virus (rAAV) Some aspects of the present disclosure relate to the use of recombinant adeno-associated virus vectors for delivery of split Cas9 proteins or split nucleobase editors into cells. The N-terminal portion of the Cas9 protein or nucleobase editor and the C-terminal portion of the Cas9 protein or nucleobase editor are delivered into the same cell by separate rAAV vectors or particles. This is because the full-length Cas9 protein or nucleobase editor exceeds the packaging limit of rAAV (approximately 4.9 kb).

[0125] Thus, in some embodiments, compositions for delivering a split Cas9 protein or a split nucleobase editor into cells (e.g., mammalian cells, human cells) are provided. In some embodiments, the compositions of the present disclosure comprise: (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein or a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein or the nucleobase editor. The rAAV particles of the present disclosure comprise an rAAV vector (i.e., a recombinant genome of an rAAV) encapsidated in a viral capsid protein.

[0126] In some embodiments, the rAAV vector comprises: (1) a heterologous nucleic acid region comprising a first or second nucleotide sequence encoding an N- or C-terminal portion of a split Cas9 protein or split nucleobase editor, in any form described herein; (2) one or more nucleotide sequences comprising a sequence (e.g., a promoter) that facilitates expression of the heterologous nucleic acid region; and (3) one or more nucleic acid regions comprising a sequence that facilitates integration of the heterologous nucleic acid region into the genome of a cell (optionally with one or more nucleic acid regions comprising sequences that facilitate expression). In some embodiments, the viral sequence that facilitates integration comprises an inverted terminal repeat (ITR). In some embodiments, the first or second nucleotide sequence encoding the N- or C-terminal portion of a split Cas9 protein or split nucleobase editor is flanked on both sides by ITR sequences. In some embodiments, the nucleic acid vector further comprises a region encoding an AAV Rep protein described herein, either within or outside the region flanked by the ITRs. The ITR sequences may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2 or AAV6.

[0127] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from: Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci USA. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 (Copyright) Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Patent Nos. 5,139,941 and 5,962,313; all of which are incorporated herein by reference. Exemplary ITR sequences are provided below. AAV2: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGCGACCAAAGGTGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (sequence number 386) AAV3: TTGGCCACTCCCTATGCGCACTCGCTCGCTCGGTGGGGCCTGGCGACCAAAGGTCGCCAGACGGATGCTTTGCACGTCGGCCCACCGAGCGAGCGAGTGCGCCATAGAGGGAGTGGCCAACTCCATCACTAGAGGTATGGC (sequence number 387) AAV5: CTCTCCCCCTGTCGCGTTCGCTCGCTCGCTGGCTCGTTTGGGGGGTGGCAGCTCAAAGAGCTGCCAGACGGCCCTCTGGCCGTCGCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCGACAGGGGGAGAGTGCCACACTCTCAAGCAAGGGGGTTTTGTA(sequence number 388) AAV6: TTGCCCACTCCCTCTATGCGCGCTCGCTCGCTCGGTGGGGCCTGCGGACCAAAGGTCCGCAGACGGCAGAGCTCTGCTCGCCCCACCGAGCGAGCGAGCGCGCATAGAGGGAGTGGGCAACTCCATCTAGGGGA(sequence number 389)

[0128] In some embodiments, the rAAV vector of the present disclosure comprises one or more regulatory elements (e.g., promoters, transcription terminators, and / or other regulatory elements) for controlling the expression of the heterologous nucleic acid region. In some embodiments, the first and / or second nucleotide sequence is operably linked to one or more (e.g., 1, 2, 3, 4, 5, or more) transcription terminators. Non-limiting examples of transcription terminators that can be used according to the present disclosure include the transcription terminators of the bovine growth hormone gene (bGH), human growth hormone gene (hGH), SV40, CW3, Φ, or combinations thereof. The efficiency of several transcription terminators was tested to determine their respective effects on the expression level of the split Cas9 protein or split nucleobase editor (see, e.g., Figure 4). In some embodiments, the transcription terminator used in the present disclosure is a bGH transcription terminator. In some embodiments, the rAAV vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE is inserted 5' of the transcription terminator.

[0129] In some embodiments, a composition comprising rAAV particles (in any form contemplated herein) further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated in a suitable pharmaceutical vehicle for administration to a human or animal subject.

[0130] Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) fats and oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH-buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols such as ethanol; and (23) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably herein.

[0131] How to use Another aspect of the present disclosure provides a method for delivering a split Cas9 protein or a split nucleobase editor into a cell to form a complete and functional Cas9 protein or nucleobase editor. For example, in some embodiments, a cell is contacted with a composition described herein (e.g., a composition comprising an AAV particle comprising a nucleotide sequence encoding a split Cas9 or a split nucleobase editor, or a nucleic acid vector comprising such a nucleotide sequence). In some embodiments, the contacting results in delivery of the nucleotide sequence into the cell, wherein the N-terminal portion of the Cas9 protein or nucleobase editor and the C-terminal portion of the Cas9 protein or nucleobase editor are expressed and linked in the cell to form a complete Cas9 protein or a complete nucleobase editor.

[0132] Split Cas9 proteins or split nucleobase editors delivered using the methods described herein preferably have comparable activity to the native Cas9 protein or nucleobase editor (i.e., the non-split protein delivered to or expressed in a cell as a whole). For example, the split Cas9 protein or split nucleobase editor retains at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) of the activity of the native Cas9 protein or nucleobase editor. In some embodiments, the split Cas9 protein or split nucleobase editor is more active (e.g., 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or more) than the activity of the native Cas9 protein or nucleobase editor.

[0133] The compositions described herein may be administered to a subject in need thereof in a therapeutically effective amount to treat and / or prevent a disease or disorder from which the subject is suffering. Any disease or disorder that can be treated and / or prevented using CRISPR / Cas9-based genome editing technology can be treated with the split Cas9 protein or split nucleobase editor described herein. It should be understood that if the nucleotide sequence encoding the split Cas9 protein or nucleobase editor does not further encode a gRNA, a separate nucleic acid vector encoding a gRNA may be administered together with the compositions described herein. Exemplary suitable diseases and disorders include, without limitation: [[The following diseases were included in the C to T editor application. Please indicate any that may be appropriate and can be treated using adenosine deaminase.] Cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR / Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013;13:653-658; and Wu et al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013;13:659-662; neither of which uses a deaminase fusion protein to correct the genetic defect); phenylketonuria—e.g., a T>C mutation at position 835 (mouse) or 240 (human) phenylalanine or a homologous residue in the phenylalanine hydroxylase gene—e.g., a T>C mutation—e.g., a T>C mutation at position 835 (mouse) or 240 (human) phenylalanine or a homologous residue in the phenylalanine hydroxylase gene—e.g., McDonald et al., Genomics. 1997;39:402-405; Bernard-Soulier syndrome (BSS)—e.g., a phenylalanine to serine mutation at position 55 or a homologous residue in platelet membrane glycoprotein IX, or a cysteine ​​to arginine mutation at residue 24 or a homologous residue (T>C mutation)—see, e.g., Noris et al., British Journal of Haematology. 1997;97:312-320, and Ali et al., Hematol. 2014;93:381-384; epidermolytic keratosis (EHK)—e.g., a leucine or homologous residue to proline mutation (counting the initiator methionine) at position 160 or 161 in keratin 1 (T>C mutation)—see, e.g., Chipev et al., Cell.1992;70:821-828; see also accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD)—e.g., a mutation of leucine or a homologous residue at position 54 or 55 in α1-antitrypsin in the processed form, or of residue 78 or a homologous residue in the unprocessed form (counting the initiator methionine) to proline (T>C mutation)—see, e.g., Poller et al., Genomics. 1993;17:740-743; see also accession number P01011 in the UNIPROT database; Charcot-Marie-Tooth type 4J disease—e.g., a mutation of isoleucine or a homologous residue at position 41 in FIG4 to threonine (T>C mutation)—see, e.g., Lenk et al., PLoS Genetics. 2011;7:e1002104; neuroblastoma (NB)—e.g., a mutation of a leucine or homologous residue at position 197 in caspase-9 to a proline (T>C mutation)—see, e.g., Kundu et al., 3 Biotech. 2013, 3:225-234; von Willebrand disease (vWD)—e.g., a mutation of a cysteine ​​or homologous residue at position 509 in the processed form of von Willebrand factor or a mutation of a cysteine ​​or homologous residue at position 1272 in the unprocessed form of von Willebrand factor to an arginine (T>C mutation)—see, e.g., Lavergne et al., Br. J. Haematol. 1992;82:66-72; see also accession number P04275 in the UNIPROT database; congenital myotonia - see, e.g., a mutation of cysteine ​​or a homologous residue at position 277 in the muscle chloride gene CLCN1 to arginine (T>C mutation) - see, e.g., Weinberger et al., The J. of Physiology.2012;590:3449-3464; hereditary renal amyloidosis—e.g., a mutation of a stop codon or homologous residue at position 78 in the processed form of apolipoprotein AII, or at position 101 in the unprocessed form, to arginine (T>C mutation)—see, e.g., Yazaki et al., Kidney Int. 2003;64:11-16; dilated cardiomyopathy (DCM)—e.g., a mutation of a tryptophan or homologous residue at position 148 in the FOXD4 gene to arginine (T>C mutation), see, e.g., Minoretti et al., Int. J. of Mol. Med. 2007;19:369-372; hereditary lymphedema—e.g., a histidine or homologous residue to arginine mutation at position 1035 in VEGFR3 tyrosine kinase (A>G mutation), see, e.g., Irrthum et al., Am. J. Hum. Genet. 2000;67:295-301; familial Alzheimer's disease—e.g., an isoleucine or homologous residue to valine mutation at position 143 in presenilin 1 (A>G mutation), see, e.g., Gallo et al., J. Alzheimer's disease. 2011;25:425-431; prion disease—e.g., a methionine or homologous residue to valine mutation at position 129 in prion protein (A>G mutation)—see, e.g., Lewis et al., J. of General Virology. 2006;87:2443-2449; chronic infantile neurological cutaneous and articular syndrome (CINCA) - e.g., a mutation of tyrosine or a homologous residue at position 570 in cryopyrin to cysteine ​​(A>G mutation) - see, e.g., Fujisawa et. al. Blood. 2007;109:2903-2911; and desmin-related cardiomyopathy (DRM) - e.g., a mutation of arginine or a homologous residue at position 120 in αβ-crystallin to glycine (A>G mutation) - see, e.g., Kumar et al., J. Biol. Chem.1999;274:24137-24141. The entire contents of all references and database entries are incorporated herein by reference.

[0134] Suitable routes of administering compositions for pain control include, without limitation, topical, subcutaneous, transdermal, intradermal, intralesional, intra-articular, intraperitoneal, intravesical, transmucosal, transgingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, periocular, intratumoral, intracerebral, parenteral, and intraventricular administration.

[0135] Compositions of the present disclosure may be administered or packaged, for example, as unit doses. The term "unit dose," when used with reference to pharmaceutical compositions of the present disclosure, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material to provide a desired therapeutic effect, in combination with a desired diluent, i.e., carrier or vehicle.

[0136] Treating a disease or disorder includes delaying the onset or progression of the disease or reducing the severity of the disease. Treating a disease does not necessarily require a curative outcome.

[0137] As used herein, "delaying" the onset of a disease means postponing, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. The delay may be of different durations depending on the history of the disease and / or the individual being treated. A method of "delaying" or reducing the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

[0138] "Onset" or "progression" of a disease refers to early signs and / or subsequent progression of the disease. Disease onset may be detectable and can be assessed using standard clinical techniques as are well known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes occurrence, recurrence, and onset.

[0139] As used herein, "onset" or "occurrence" of a disease includes initial onset and / or recurrence. Depending on the type of disease or site of disease to be treated, conventional methods known to those skilled in the art of medicine may be used to administer the isolated polypeptide or pharmaceutical composition to a subject.

[0140] kit The compositions of the present disclosure may be assembled into a kit. In some embodiments, the kit includes a nucleic acid vector for expressing a nucleobase editor described herein. In some embodiments, the kit further includes an appropriate guide nucleotide sequence (e.g., gRNA) or a nucleic acid vector for expressing such a guide nucleotide sequence to target the Cas9 protein or nucleobase editor to a desired target sequence.

[0141] The kits described herein may include one or more containers containing the components for carrying out the methods described herein, and optionally instructions for use. Any of the kits described herein may further include components required for carrying out assay methods. Where appropriate, each component of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., as a dry powder). In some cases, some of the components may be reconstituted or otherwise processed (e.g., into an active form), for example, by adding a suitable solvent or other species (e.g., water), which may or may not be provided with the kit.

[0142] In some embodiments, the kit may include instructions and / or promotional materials for use of the provided components. As used herein, "instructions" can define the components of the instructions and / or promotional materials and typically include written instructions on or associated with the packaging of the present disclosure. Instructions may also include oral or electronic instructions provided in any format, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., so that the user clearly recognizes that the instructions should be associated with the kit. Written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, and may also reflect agency approval of the manufacture, use, or sale for administration to animals. As used herein, "promoted" includes all methods of doing business, including educational methods, hospital and other clinical instructions, scientific research, drug discovery or development, academic research, pharmaceutical activities including pharmaceutical sales, and any advertising or other promotional activities (including written, oral, and electronic communications), related to the present disclosure. Additionally, the kit may include other components, as described herein, depending on the particular application.

[0143] The kit may contain any one or more of the components described herein in one or more containers.The components may be prepared aseptically, packaged in syringes, and transported under refrigeration.Alternatively, it may be contained in other vials or other containers for storage.A second container may contain other components that are prepared aseptically.Alternatively, the kit may contain the active ingredients that are premixed and transported in vials, tubes, or other containers.

[0144] The kit may have a variety of forms, such as a blister pouch, shrink-wrapped pouch, vacuum-sealed pouch, sealed thermoformed tray, or similar pouch or tray form, with the accessories loosely packaged within the pouch, one or more tubes, containers, boxes, or bags. The kit can be sterilized after the accessories are added, allowing the individual accessories in the container to be opened separately. The kit can be sterilized using any suitable sterilization technique, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. Depending on the specific application, the kit may also include other components, such as containers, cell culture media, salts, buffering agents, reagents, syringes, needles, fabrics such as gauze, etc., such as disinfectants, disposable gloves, and materials for applying or removing support materials for agents prior to administration.

[0145] host cell Cells that may contain any of the compositions described herein include prokaryotic and eukaryotic cells. The methods described herein are used to deliver a Cas9 protein or nucleobase editor into eukaryotic cells (e.g., mammalian cells, such as human cells). In some embodiments, the cells are in vitro (e.g., cultured cells). In some embodiments, the cells are in vivo (e.g., a subject, such as a human subject). In some embodiments, the cells are ex vivo (e.g., isolated from a subject and can be administered back to the same or a different subject).

[0146] Mammalian cells of the present disclosure include human cells, primate cells (e.g., Vero cells), rat cells (e.g., GH3 cells, OC23 cells), or mouse cells (e.g., MC3T3 cells). A variety of human cell lines exist, including, but not limited to, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma), and Saos-2 (bone cancer) cells. In some embodiments, the rAAV vector is delivered into human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, the rAAV vector is delivered into stem cells (e.g., human stem cells), such as pluripotent stem cells (e.g., human pluripotent stem cells, including human-derived pluripotent stem cells (hiPSCs)). Stem cells refer to cells that have the ability to divide in culture for an indefinite period of time and generate specialized cells. Pluripotent stem cells refer to a type of stem cell that can differentiate into all tissues of an organism but cannot alone maintain the development of a complete organism. Human-derived pluripotent stem cells refer to somatic (e.g., adult or adult) cells that have been reprogrammed into an embryonic stem cell-like state by forcing the expression of genes and factors that are important for maintaining the defining characteristics of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126(4):663-76, 2006; incorporated herein by reference). Human-derived pluripotent stem cells express stem cell markers and can give rise to the characteristics of cells of all three lung lobes (ectoderm, endoderm, and mesoderm).

[0147] Further non-limiting examples of cell lines that can be used in accordance with the present disclosure include: 293-T, 293-T, 3T3, 4T1, 721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Cal-27, CGR8, CHO, CML T1, CMT, COR-L23, COR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23, COS-7, COV-434, CT26, D17, DH82, DU145, Du CaP, E14Tg2a, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepa1c1c7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812, KYO1, LNCap, Ma-Mel 1, 2, 3...48, MC-38, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR / 0.2R, MRC5, MTD-1A, MyEnd, NALM-1, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NW-145, OPCN / OPCT Peer, PNT-1A / PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA / RMAS, S2, Saos-2 cells, Sf21, Sf9, SiHa, SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1 and YAR cells.

[0148] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present disclosure to its fullest extent. The following specific aspects are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0149] example In order that the invention described herein may be more fully understood, the following examples are set forth: The synthetic examples described herein are provided to illustrate the compounds and methods provided herein and should not be construed in any way as limiting the scope thereof.

[0150] Example 1: Amino acid sequences of Cas9 proteins and nucleobase editors Non-limiting examples of suitable Cas9 proteins and variants, as well as nucleobase editors and variants, are provided. The present disclosure provides Cas9 variants, e.g., Cas9 proteins from one or more organisms, which may include one or more mutations (e.g., to generate dCas9 or Cas9 nickase). In some embodiments, one or more of the amino acid residues of a Cas9 protein, identified below with an asterisk, may be mutated. In some embodiments, the D10 and / or H840 residues of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, are mutated. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is mutated to any amino acid residue other than D. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is mutated to A. In some embodiments, residue H840 of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is H. In some embodiments, residue H840 of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is mutated to any amino acid residue other than H. In some embodiments, residue H840 of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is mutated to A. In some embodiments, residue D10 of the amino acid sequence provided in SEQ ID NO: 1, or the corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 2-275 and 394-397, is D.

[0151] Multiple Cas9 sequences from various species were aligned to determine whether the corresponding homologous amino acid residues of D10 and H840 in SEQ ID NO: 1 could be identified in other Cas9 proteins, allowing for the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues. Alignment was performed using NCBI's Constraint-based Multiple Alignment Tool (COBALT, accessible at st-va.ncbi.nlm.nih.gov / tools / cobalt) with the following parameters: Gap penalties -11, -1; End-Gap penalties -5, -1. CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on. Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. S. pyogenes Cas9 wild type (NCBI reference sequence: NC_002737.2, Uniprot reference sequence: Q99ZW2) S. pyogenes dCas9 (D10A and H840A) S. pyogenes Cas9 nickase (D10A) VRER-nCas9(D10A / D1135V / G1218R / R1335E / T337R)S. pyogenes Cas9nickerbocker VQR-nCas9(D10A / D1135V / R1335Q / T1337R) S. pyogenes Cas9 nickase EQR-nCas9(D10A / D1135E / R1335Q / T1337R) S. pyogenes Cas9 nickase KKH-nCas9(D10A / E782K / N968K / R1015H) S. aureus Cas9 nickase Streptococcus thermophilus CRISPR1 Cas9(St1Cas9)ニッカーゼ(D9A) Streptococcus thermophilus CRISPR3 Cas9(St3Cas9)ニッカーゼ(D10A) S. aureus Cas9 wild type S. aureus Cas9 nickase (D10A) Streptococcus thermophilus wild-type CRISPR3 Cas9 (St3Cas9) Streptococcus thermophilus CRISPR1 Cas9 wild type (St1Cas9) CasX from Sulfolobus islandicus (strain REY15A). MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG (SEQ ID NO: 14) CasY from Sulfolobus islandicus (strain REY15A). MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG (SEQ ID NO: 15) Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bold and underlined)

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[0152] An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are as follows: Sequence 1 (S1): SEQ ID NO: 1|WP_010922251|gi 499224711|Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes]; Sequence 2 (S2): SEQ ID NO: 27|WP_039695303|gi 746743737|Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus gallolyticus]; Sequence 3 (S3): SEQ ID NO: 28|WP_045635197|gi 782887988|Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus mitis]; Sequence 4 (S4): SEQ ID NO: 29|5AXW_A|gi 924443546|Staphylococcus aureus Cas9. The HNH domain (bold and underlined) and RuvC domain (boxed) are identified for each of the four sequences. Amino acid residues 10 and 840 in S1 and homologous amino acids in the aligned sequences are identified by an asterisk after the respective amino acid residue.

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[0153] Alignment refers to the identification of amino acid sequences and residues homologous to a reference Cas9 amino acid sequence or residue across Cas9 sequence variants, including Cas9 sequences from different species, by identifying amino acid sequences or residues that align with the reference sequence or residues using alignment programs and algorithms known in the art, including, but not limited to, Cas9 sequences from different species. The present disclosure provides Cas9 variants in which one or more of the amino acid residues identified by asterisks in SEQ ID NOS: 1 and 27-29 (e.g., S1, S2, S3, and S4, respectively) have been mutated as described herein. Residues D10 and H840 in Cas9 of SEQ ID NOS: 1, which correspond to the residues identified by asterisks in SEQ ID NOS: 1 and 27-29, are referred to herein as "homologous" or "corresponding" residues. Such homologous residues can be identified by identifying sequences or residues that align with a reference sequence or residue, for example, by sequence alignment, as described above. Similarly, mutations in the Cas9 sequence corresponding to those identified in SEQ ID NO:1, e.g., mutations at residues 10 and 840 in SEQ ID NO:1, are referred to herein as "homologous" or "corresponding" mutations. For example, for the four aligned sequences, the mutations corresponding to the D10A mutation in SEQ ID NO:1 (S1) are D11A for S2, D10A for S3, and D13A for S4; the corresponding mutations for H840A in SEQ ID NO:1 (S1) are H850A for S2, H842A for S3, and H560A for S4.

[0154] A total of 250 Cas9 sequences from different species are provided (SEQ ID NOS: 1 and 27-275). The amino acid residues corresponding to residues 10 and 840 in SEQ ID NOS: 1 can be identified in the same manner as outlined above. All of these Cas9 sequences can be used in accordance with the present disclosure. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

[0155] Non-limiting examples of suitable cytosine deaminase domains are provided. Human AID MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 276) Mouse AID MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF (SEQ ID NO: 277) Inu AID MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 278) Cow AID MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 279) Mouse APOBEC-3 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECA EQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTK GLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 280) Rat APOBEC-3 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECA EQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTK GLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 281) Rhesus APOBEC-3G MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEMRFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRCANSVATFL AKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNK PWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO: 282) (italics: nucleic acid editing domain; underline: cytoplasmic localization signal) Chimpanzee APOBEC-3G MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 283) Green monkey APOBEC-3G MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI (SEQ ID NO: 284) Human APOBEC-3G MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 285) Human APOBEC-3F MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE (SEQ ID NO: 286) Human APOBEC-3B MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 287) Human APOBEC-3C: MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ (SEQ ID NO: 288) Human APOBEC-3A: MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (SEQ ID NO: 289) Human APOBEC-3H: MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV (SEQ ID NO: 290) Human APOBEC-3D MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (SEQ ID NO: 291) Human APOBEC-1 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 292) Mouse APOBEC-1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK (SEQ ID NO: 293) Rat APOBEC-1 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 294) Lamprey CDA1 (pmCDA1) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 295) Human APOBEC3G D316R_D317R MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO: 296) Human APOBEC3G chain A MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 297) Human APOBEC3G chain A D120R_D121R MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ (SEQ ID NO: 298)

[0156] Non-limiting exemplary uracil glycosylase inhibitor sequences are provided. Bacteriophage PBS2 (Bacteriophage PBS2) uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 299) Erwinia tasmaniensis SSB (thermostable single-stranded DNA binding protein) MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETKEKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTTEVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGGAQQQARPQQQPQQNNAPANNEPPIDFDDDIP (SEQ ID NO: 300) UdgX (binds to uracil in DNA but does not excise it) MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMIGEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTRAAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGNDFRVTQHRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDDLRVAADVRP (SEQ ID NO: 301) UDG (catalytically inactive human UDG, which binds to but does not excise uracil in DNA) MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKKAPAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKKHLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVILGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGDLSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELLQKSGKKPIDWKEL (SEQ ID NO: 302)

[0157] Non-limiting examples of C to T nucleobase editors are provided. His6-rAPOBEC1-XTEN-dCas9 (SEQ ID NO: 303) for expression in Escherichia coli rAPOBEC1-XTEN-dCas9-NLS (SEQ ID NO: 304) for mammalian expression hAPOBEC1-XTEN-dCas9-NLS (SEQ ID NO: 305) for mammalian expression rAPOBEC1-XTEN-dCas9-UGI-NLS (SEQ ID NO: 306) rAPOBEC1-XTEN-Cas9 Nickase-UGI-NLS (BE3, SEQ ID NO: 307) pmCDA1-XTEN-dCas9-UGI (bacteria) (SEQ ID NO: 308) pmCDA1-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 309) huAPOBEC3G-XTEN-dCas9-UGI(bacteria) (SEQ ID NO: 310) huAPOBEC3G-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 311) huAPOBEC3G(D316R_D317R)-XTEN-nCas9-UGI-NLS (mammalian construct) (SEQ ID NO: 312) High-fidelity nucleobase editor (SEQ ID NO: 313) rAPOBEC1-XTEN-SaCas9n-UGI-NLS)

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[0158] Non-limiting examples of evolved adenosine deaminases that accept DNA as a substrate are provided.

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[0159] Non-limiting examples of A to G nucleobase editors are provided. ecTadA(wt)-XTEN-nCas9-NLS (SEQ ID NO: 323) ecTadA(D108N)-XTEN-nCas9-NLS: (Mammalian construct, active on DNA, A to G editing, SEQ ID NO: 324) ecTadA(D108G)-XTEN-nCas9-NLS: (Mammalian construct, active on DNA, A to G editing, SEQ ID NO: 325) ecTadA(D108V)-XTEN-nCas9-NLS: (Mammalian construct, active on DNA, A to G editing, SEQ ID NO: 326) ecTadA(D108N)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor, SEQ ID NO: 327) ecTadA(D108G)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor, SEQ ID NO: 328): ecTadA(D108V)-XTEN-nCas9-UGI-NLS (BE3 analog of A to G editor, SEQ ID NO: 329): ecTadA(D108N)-XTEN-dCas9-UGI-NLS (mammalian cells, BE2 analog of A to G editor, SEQ ID NO: 330): ecTadA(D108G)-XTEN-dCas9-UGI-NLS (mammalian cells, BE2 analog of A to G editor, SEQ ID NO: 331): ecTadA(D108V)-XTEN-dCas9-UGI-NLS (mammalian cells, BE2 analog of A to G editor, SEQ ID NO: 332): ecTadA(D108N)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyl adenosine glycosylase (SEQ ID NO: 333) ecTadA(D108G)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyl adenosine glycosylase (SEQ ID NO: 334) ecTadA(D108V)-XTEN-nCas9-AAG(E125Q)-NLS-cat. alkyl adenosine glycosylase (SEQ ID NO: 335) ecTadA(D108N)-XTEN-nCas9-EndoV(D35A)-NLS: containing cat. endonuclease V (SEQ ID NO: 336) ecTadA(D108G)-XTEN-nCas9-EndoV(D35A)-NLS: cat. Endonuclease V (SEQ ID NO: 337) ecTadA(D108V)-XTEN-nCas9-EndoV(D35A)-NLS: cat. Endonuclease V (SEQ ID NO: 338) Variant ecTadA(H8Y_D108N_N127S)-XTEN-dCas9 (SEQ ID NO: 339) resulting from the first round of evolution (in bacteria) Enriched variant ecTadA(H8Y_D108N_N127S_E155X)-XTEN-dCas9 from the second round of evolution (in bacteria); X = D, G, or V (SEQ ID NO: 340) pNMG-160: ecTadA(D108N)-XTEN-nCas9-GGS-AAG*(E125Q)-GGS-NLS (SEQ ID NO: 341) pNMG-161:ecTadA(D108N)-XTEN-nCas9-GGS-EndoV*(D35A)-GGS-NLS (SEQ ID NO: 342) pNMG-371: ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-SGGS-SGGS-XTEN-SGGS-SGGS- ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-SGGS-SGGS-XTEN-SGGS-SGGS-nCas9-SGGS-NLS (SEQ ID NO: 458) pNMG-616 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F _K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 459) pNMG-624 amino acid sequence: ecTadA (wild type)-32 a.a. linker-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24 a.a. linker_nCas9_SGGS_NLS (SEQ ID NO: 460) pNMG-476 amino acid sequence (evolution #3 heterodimer, wt TadA + TadA evo #3 mutation): ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 461) pNMG-477 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 462) pNMG-558 amino acid sequence: ecTadA (wild type)-32 a.a. linker-ecTadA (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-24 a.a. linker_nCas9_SGGS_NLS (SEQ ID NO: 463) pNMG-576 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 464) pNMG-577 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 465) pNMG-586 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 466) pNMG-588 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 467) pNMG-620 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 468) pNMG-617 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 469) pNMG-618 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 470) pNMG-620 amino acid sequence: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_GGS_NLS (SEQ ID NO: 471) pNMG-621 amino acid sequence: ecTadA (wild type)-32 a.a. linker-ecTadA (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24 a.a. linker_nCas9_GGS_NLS (SEQ ID NO: 472) pNMG-622 amino acid sequence: ecTadA (wild type)-32 a.a. linker-ecTadA (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)-24 a.a. linker_nCas9_GGS_NLS (SEQ ID NO: 473) pNMG-623 amino acid sequence: ecTadA (wild type)-32 a.a. linker-ecTadA (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-24 a.a. linker_nCas9_GGS_NLS (SEQ ID NO: 474) ABE6.3 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 475) ABE7.8 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 476) ABE7.9 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 477) ABE7.10 ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS ABE6.4: ecTadA (wild type)-(SGGS)2-XTEN-(SGGS)2-ecTadA(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)-(SGGS)2-XTEN-(SGGS)2_nCas9_SGGS_NLS (SEQ ID NO: 480)

[0160] Example 2: AAV delivery of split nucleobase editors This study was designed to demonstrate that a nucleobase editor can be delivered by recombinant AAV (rAAV) in two sections, which can be joined in cells via protein splicing to form a complete and active nucleobase editor. Various elements of the rAAV construct were tested for expression and activity of the optimized nucleobase editor.

[0161] Recombinant AAV (rAAV) is widely used for transgene delivery. A transgene is inserted into the AAV genome between inverted terminal repeats (ITRs) and packaged into AAV viral particles used to transduce host cells (e.g., mammalian cells, human cells). However, there is a limit (typically about 4.9 kilobases) to the size of the transgene that can be packaged in rAAV. A nucleic acid encoding a nucleobase editor (e.g., cytosine deaminase-dCas9-UGI) typically exceeds the packaging limit of rAAV. As described herein, a nucleic acid encoding a nucleobase editor was split (see FIG. 1A), and each section was packaged into a separate rAAV particle. The two sections of the nucleobase editor can be delivered to a cell and ligated to form a complete nucleobase editor through protein splicing (e.g., mediated by an intein such as DnaE intein; see FIG. 1C). The ligated complete nucleobase editor was active in editing the target base (see Figure 1B). rAAV constructs encoding split nucleobase editors were tested in different cell lines, such as U118 and HEK293T, and were active in editing the target base (see Figures 3A-3B and 5A-5B).

[0162] Various transcription terminators and nuclear localization signals (NLSs) were tested in rAAV constructs to optimize the expression and activity of the nucleobase editors (see Figures 4, 6, and 7).

[0163] Example 3: Editing the DNMT1 gene using an AAV encoding a split nucleobase editor in mouse neurons This study was designed to test the base editing activity of AAVs encoding split nucleobase editors in vivo. A split nucleobase editor, as shown in Figure 1A, was used. The amino acid sequence of the linker between the dCas9 domain and the deaminase domain is SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 384). A guide RNA targeting a well-characterized site in the DNMT1 gene was selected. It was predicted that cells would be able to tolerate editing. These experiments aimed to determine whether AAVs encoding split base editors could edit gene loci in vivo in several cell types, including primate neurons.

[0164] In one experiment, an AAV encoding a split nucleobase editor and a guide RNA targeting DNMT1 were used to transduce isolated mouse cortical neurons 2 days after isolation and culture. Neurons were collected 16 days after transduction, and the DNMT1 gene was sequenced (Figure 8A) to determine editing efficiency and off-target effects. We detected a 17.34% editing efficiency (C-to-T edits, darker gray in Figure 8B), while detecting only 0.82% of undesired edits (C-to-G or C-to-A changes, lighter gray in Figure 8B).

[0165] In a separate experiment, cultured mouse Neuro-2 cells were transduced with an AAV vector encoding a split nucleobase editor and a guide RNA targeting DNMT1, or with DNA and guide RNA encapsulated in lipids encoding the nucleobase editor, thereby allowing a direct comparison of the editing efficiencies using different delivery methods of the nucleobase editor (Figure 9A). An editing efficiency of 5.96% (C to T editing, dark gray in Figure 9B) was observed for AAV encoding the split nucleobase editor, while an editing efficiency of 27.3% (C to T editing, dark gray in Figure 9B) was observed for DNA transfected with lipids encoding the nucleobase editor. The amount of undesired products was 0.15% for AAV encoding the split nucleobase editor and 1.3% for DNA transfected with lipids encoding the nucleobase editor (C to G or C to A changes, lighter gray in Figure 9B).

[0166] Equivalents and Scope In the claims, the articles "a," "an," and "the" can mean one or more than one, unless contradictory or otherwise clear from context. A claim or description including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless contradictory or otherwise clear from context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of a group are present in, employed in, or otherwise relevant to a given product or process.

[0167] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same base claim. When elements are present in a list, e.g., in Markush format, each subgroup of elements is also disclosed, and any element can be removed from the group. In general, when the invention or aspects of the invention are referred to as including particular elements and / or features, it should be understood that certain embodiments or aspects of the invention consist of or consist essentially of such elements and / or features. For simplicity's sake, these embodiments have not been specifically recited in words herein.

[0168] It is also noted that the terms "comprising" and "containing" are intended to be open, permitting the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated, or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​presented as ranges can assume any specific value or subrange of the ranges described in different aspects of the invention, to the tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0169] This application references various issued patents, published patent applications, academic articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, the specification shall control. Furthermore, any particular aspect of the present invention that constitutes prior art may be expressly excluded from any one or more of the claims. Because such aspects are deemed known to those skilled in the art, they may be excluded even if the exclusion is not explicitly set forth herein. Any particular aspect of the present invention may be excluded from any claim for any reason, regardless of whether it relates to the existence of prior art.

[0170] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications may be made to this description without departing from the spirit or scope of the invention as defined in the following claims.

Claims

1. (i) a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein; A composition comprising: wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal; The composition.

2. 2. The composition of claim 1, wherein the N-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, corresponding to amino acids 1-573 or 1-637 of SEQ ID NO:

1.

3. 3. The composition of claim 1 or 2, wherein the C-terminal portion of the Cas9 protein comprises a portion of any one of SEQ ID NOs: 1-275 and 394-397, corresponding to amino acids 574-1368 or 638-1368 of SEQ ID NO:

1.

4. The composition of any one of claims 1 to 3, wherein intein-N comprises an amino acid sequence set forth in any one of SEQ ID NOs: 350-351 and 354-355.

5. The composition of any one of claims 1 to 4, wherein intein-C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 352-353 and 356-357.

6. 6. The composition of any one of claims 1 to 5, wherein the first nucleotide sequence or the second nucleotide sequence further comprises nucleotides encoding a guide RNA (gRNA) operably linked to a promoter.

7. The composition of any one of claims 1 to 6, wherein the first nucleotide sequence or the second nucleotide sequence further comprises a transcription terminator.

8. The composition of claim 7, wherein the transcription terminator is a transcription terminator from the bGH gene, the hGH gene, or the SV40 gene.

9. The composition of claim 8, wherein the transcription terminator is a transcription terminator from the bGH gene.

10. 10. The composition of any one of claims 1 to 9, wherein the first nucleotide sequence or the second nucleotide sequence further comprises a Woodchuck Hepatitis post-transcriptional regulatory element (WPRE) inserted 5' to the transcription terminator.

11. 11. The composition of any one of claims 1 to 10, wherein the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of KRPAATKKAGQAKKKK (SEQ ID NO: 344), KKTELQTTNAENKTKKL (SEQ ID NO: 345), KRGINDRNFWRGENGRKTR (SEQ ID NO: 346), and RKSGKIAAIVVKRPRK (SEQ ID NO: 347).

12. The composition of claim 11, wherein the bipartite nuclear localization signal comprises the amino acid sequence set forth in SEQ ID NO:

344.

13. 13. The composition of any one of 1-12, wherein the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), and wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the N-terminus of the N-terminal portion of the Cas9 protein.

14. 13. The composition of any one of claims 1 to 12, wherein the Cas9 protein is a catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9), and wherein the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a nucleobase modifying enzyme fused to the C-terminus of the C-terminal portion of the Cas9 protein.

15. 15. The composition of claim 13 or 14, wherein the nucleobase modifying enzyme is a deaminase.

16. The composition of claim 15, wherein the deaminase is a cytosine deaminase.

17. 16. The composition of claim 15, wherein the deaminase is adenosine deaminase.

18. The composition of any one of claims 14 to 17, wherein the second nucleotide sequence of (ii) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) at the 3' end of the second nucleotide sequence.

19. The composition of any one of claims 13 to 17, wherein the first nucleotide sequence of (i) further comprises a nucleotide sequence encoding a uracil glycosylase inhibitor (UGI) at the 5' end of the first nucleotide sequence.

20. The composition of claim 18 or 19, wherein the UGI comprises an amino acid sequence of SEQ ID NO: 299-302.

21. The composition of any one of claims 1 to 20, wherein the first and second nucleotide sequences are on different vectors.

22. 22. The composition of claim 21, wherein each of the different vectors is a recombinant adeno-associated virus (rAAV) genome.

23. The composition of claim 22, wherein each vector is packaged in an rAAV particle.

24. (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a Cas9 protein fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the Cas9 protein; A composition comprising: wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal; The composition.

25. A cell comprising the composition of any one of claims 1 to 24.

26. 26. The cell of claim 25, wherein the N-terminal portion of the Cas9 protein and the C-terminal portion of the Cas9 protein are linked together to form the Cas9 protein.

27. 27. The cell of claim 25 or 26, which is a prokaryotic cell.

28. 28. The cell of claim 27 which is a bacterial cell.

29. 27. The cell of claim 25 or 26, which is a eukaryotic cell.

30. 30. The cell of claim 29, which is a yeast cell, a plant cell, or a mammalian cell.

31. 30. The cell of claim 29, which is a human cell.

32. A kit comprising the composition of any one of claims 1 to 24.

33. (i) a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second nucleotide sequence encoding an intein-C fused to the N-terminus of the C-terminal portion of the nucleobase editor. A composition comprising:

34. 34. The composition of claim 33, wherein the intein-N comprises an amino acid sequence set forth in any one of SEQ ID NOs: 350-351 and 354-355.

35. The composition of claim 33 or 34, wherein the intein-C comprises an amino acid sequence set forth in any one of SEQ ID NOs: 352-353 and 356-357.

36. 36. The composition of any one of claims 33 to 35, wherein the first nucleotide sequence or the second nucleotide sequence further comprises nucleotides encoding a guide RNA (gRNA) operably linked to a promoter.

37. The composition of any one of claims 33 to 36, wherein the first nucleotide sequence or the second nucleotide sequence further comprises a transcription terminator.

38. 38. The composition of claim 37, wherein the transcription terminator is a transcription terminator from the bGH gene, the hGH gene, or the SV40 gene.

39. The composition of claim 38, wherein the transcription terminator is from the bGH gene.

40. 40. The composition of any one of claims 33 to 39, wherein the first nucleotide sequence or the second nucleotide sequence further comprises a Woodchuck Hepatitis post-transcriptional regulatory element (WPRE) inserted 5' to the transcription terminator.

41. The composition of any one of claims 33 to 40, wherein the first nucleotide sequence or the second nucleotide sequence is operably linked to a nucleotide sequence encoding at least one bipartite nuclear localization signal.

42. 42. The composition of any one of claims 33-41, wherein the bipartite nuclear localization signal comprises an amino acid sequence selected from the group consisting of KRPAATKKAGQAKKKK (SEQ ID NO:344), KKTELQTTNAENKTKKL (SEQ ID NO:345), KRGINDRNFWRGENGRKTR (SEQ ID NO:346), and RKSGKIAAIVVKRPRK (SEQ ID NO:347).

43. The composition of claim 42, wherein the bipartite nuclear localization signal comprises the amino acid sequence set forth in SEQ ID NO:

344.

44. 44. The composition of any one of claims 33-43, wherein the nucleobase editor comprises a cytosine deaminase fused to the N-terminus of a catalytically inactive Cas9 or Cas9 nickase.

45. The composition of claim 44, wherein the cytosine deaminase is selected from the group consisting of APOBEC1, APOBEC3, AID and pmCDA1.

46. 46. ​​The composition of claims 44 or 45, wherein the nucleobase editor further comprises a uracil glycosylase inhibitor (UGI).

47. 47. The composition of claim 46, wherein the UGI comprises an amino acid sequence of SEQ ID NOs: 299-302.

48. The composition of any one of claims 33 to 47, wherein the first nucleotide sequence and the second nucleotide sequence are on different vectors.

49. 49. The composition of claim 48, wherein each of the different vectors is a recombinant adeno-associated virus (rAAV) genome.

50. 50. The composition of claim 49, wherein the vector is packaged in a rAAV particle.

51. (i) a first recombinant adeno-associated virus (rAAV) particle comprising a first nucleotide sequence encoding an N-terminal portion of a nucleobase editor fused at its C-terminus to an intein-N; and (ii) a second recombinant adeno-associated virus (rAAV) particle comprising a second nucleic acid encoding an intein-C fused to the N-terminus of the C-terminal portion of the nucleobase editor; A composition comprising:

52. A cell comprising the composition of any one of claims 33 to 51.

53. 53. The cell of Claim 52, wherein the N-terminal portion of the nucleobase editor and the C-terminal portion of the nucleobase editor are linked together to form the nucleobase editor.

54. 54. The cell of claim 52 or 53, which is a prokaryotic cell.

55. 55. The cell of claim 54 which is a bacterial cell.

56. 54. The cell of claim 52 or 53, which is a eukaryotic cell.

57. 57. The cell of claim 56 which is a yeast cell, a plant cell, or a mammalian cell.

58. 57. The cell of claim 56, which is a human cell.

59. A kit comprising the composition of any one of claims 33 to 51.

60. 25. A method comprising contacting a cell with the composition of any one of claims 1 to 24, wherein the contacting results in delivery of a first nucleotide sequence and a second nucleotide sequence into the cell, and wherein an N-terminal portion of the Cas9 protein and a C-terminal portion of the Cas9 protein are linked to form the Cas9 protein.

61. 52. A method comprising contacting a cell with the composition of any one of claims 33-51, wherein the contacting results in delivery of a first nucleotide sequence and a second nucleotide sequence into the cell, and wherein an N-terminal portion of the nucleobase editor and a C-terminal portion of the nucleobase editor are linked to form the nucleobase editor.

62. A method comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to any one of claims 1-24 and 33-51.

63. 63. The method of claim 62, wherein the subject has a disease or disorder.

64. 64. The method of claim 63, wherein the disease or disorder is selected from the group consisting of cystic fibrosis, phenylketonuria, epidermolytic keratosis (EHK), chronic obstructive pulmonary disease (COPD), Charcot-Marie-Tooth type 4J disease, neuroblastoma (NB), von Willebrand disease (vWD), congenital myotonia, hereditary renal amyloidosis, dilated cardiomyopathy, hereditary lymphedema, familial Alzheimer's disease, prion disease, chronic infantile neurological cutaneous and articular syndrome (CINCA), and desmin-related cardiomyopathy (DRM).

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